Clinically intelligent diagnostic devices and methods

ABSTRACT

The invention relates to the clinically intelligent design of diagnostic devices (such as microarrays) and methods of making and using such devices in differential diagnoses of specific clinical symptoms or sets of symptoms. In one aspect, the devices include various probes used to perform parallel screening of a number of analytes. The probes are clustered on the devices based on known clinical presentations of symptoms associated with specific diseases and disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional PatentApplication Ser. Nos. 60/253,284, filed on Nov. 27, 2000; 60/287,994,filed on May 1, 2001; and 60/308,870, filed on Jul. 30, 2001, which areall incorporated herein by references in their entirety.

TECHNICAL FIELD

This invention relates to medical diagnosis.

BACKGROUND

It is common for patients to seek the advice of a physician whenexperiencing discomfort. However, patients seldom present physicianswith a diagnosis already made; instead, they present one or moresymptoms. Selecting the most probable diagnosis from a list ofalternatives (hypotheses) is a process called differential diagnosis.Certain signs or symptoms can suggest a specific disease etiology.However, patients typically present physicians with clinical symptomsthat are confounding, which make diagnosis based on only the symptomsvery difficult. The physician has the daunting task of determining whichof a number of principal etiologies is responsible for the discomfortthe patient is experiencing. This is also important because selecting aspecific diagnosis has implications for the treatment plan and therapy.The symptoms prompt the physician to gather information through history,physical examination and, most importantly, diagnostic tests identifyingclinical findings that suggest explanations for the symptom(s).

Thus, diagnostic tests, both performed at a laboratory and at thepoint-of-care (POC), are an integral part of the health care system.Such tests play a central role in all aspects of patient care, includingdisease-diagnosis, monitoring progression of therapy, as well asscreening for health and infection. Molecular diagnostics tests (such asin vitro diagnostic (IVD) tests) are especially useful, as they pinpointthe exact cause of a particular clinical manifestation and thus help thephysician to make a diagnosis and then prescribe the right treatment andtherapy.

Currently, the diagnostic testing process is very tedious,time-consuming, cumbersome, and slow. This is because a number ofdifferent tests often have to be performed for a given symptom and eachof these tests is performed individually. Moreover, because laboratoriesare constantly updating and adding new tests that facilitate medicaldiagnosis, physicians regularly confront dilemmas when ordering andinterpreting these tests. Over the last two decades, the total number ofclinical tests and the types of different tests available to physicianshas grown exponentially. These advances in modern clinical laboratorymedicine, though enormously helpful, also create new problems. Thesetests are often not user friendly, and increase costs in an alreadyheavily burdened health care system. In common practice, physicians alsocomplain about the delay in the processing of tests at the laboratoriesthus delaying accurate patient diagnosis. Additionally, many tests arenot available at all, are available at only one or two testingsites/laboratories, or are known only to specialists.

Effective management of diseases requires an awareness of the fullspectrum of etiologies and their possible complications. Sometimes theinitially chosen set of tests present results that are not clear, whichprecludes an accurate diagnosis. The nature and relatively non-specificsymptoms of the disease can make a proper diagnosis challenging. In suchcases more tests are performed, which are run in an iterative andsequential fashion. Thus, testing slows down the entire process ofpatient care and treatment, which is costly, and is detrimental to thepatient's health and treatment plans.

SUMMARY

The present invention relates to the clinically intelligent design ofdiagnostic devices (such as microarrays) and the methods of making andusing such devices in differential analyses/diagnoses of specificclinical symptoms or sets of symptoms. In one aspect, the devicesinclude various probes used to perform parallel screening of a number ofanalytes. The probes are clustered on the devices based on knownclinical presentations of symptoms associated with specific diseases anddisorders. In another aspect, the devices are used to perform parallelscreening of a number of clinically associated analytes, such as knownblood-borne pathogens and antibodies. In yet another aspect, thesedevices are used to perform parallel screening of analytes found inagricultural, forensic, veterinary, and other samples.

In general, the invention relates to a method of determining a cause ofone or more medical symptoms exhibited by a subject by (a) obtaining abiological sample from the subject; (b) obtaining an array of differentprobes or different sets of probes, wherein each probe or set of probesselectively interacts with a target associated with a different knowncause of the one or more medical symptoms; (c) applying the biologicalsample to the probes in the array under conditions that enable all ofthe probes to selectively interact with any targets in the biologicalsample; (d) detecting interactions; and (e) analyzing interactions todetermine a cause of the one or more medical symptoms. In this method,the array of probes or sets of probes can be arranged on a planarsubstrate. The target can be a nucleic acid, peptide, polypeptide,protein, antibody, antigen, small organic molecule, inorganic molecule,enzyme, or polysaccharide. All of the probes in the array can bepolypeptides, e.g., antibodies, antigens, enzymes, zinc-finger bindingproteins, minor-groove binders, transcriptional factors, combinationsthereof, or chimeras thereof.

In these methods, the subject can be a plant or animal, or a humanpatient or a deceased human. In certain embodiments, the probes can beexpressed on the surface of genetically modified cells, and the probescan selectively interact with a target by specifically binding to thetarget to form a complex. In certain embodiments, the array of probescan include a first probe that selectively interacts with a targetassociated with an infectious disease caused by a bacteria, virus, orfungus, and a second, different probe selectively interacts with atarget associated with a genetic cause. The array of probes can alsoinclude probes that assay for the absence of a causative agent of one ormore medical symptoms.

In another aspect, the invention features a method of determining thesusceptibility of a subject to a cause of one or more medical symptoms,by: (a) obtaining a biological sample from the subject; (b) obtaining anarray of different probes or different sets of probes, wherein eachprobe or set of probes selectively interacts with a target associatedthe susceptibility of the subject to a different cause of the one ormore medical symptoms; (c) applying the biological sample to the probesin the array under conditions that enable all of the probes toselectively interact with any targets in the biological sample; (d)detecting interactions; and (e) analyzing interactions to determine thesusceptibility of the subject to a cause of the one or more medicalsymptoms.

In the new methods, all of the probes can be designed to selectivelyinteract with their respective targets under the same conditions.

In another aspect, the invention also includes a method of determining acause of one or more medical symptoms in a subject and assessing thesuitability of one or more therapeutic agents to treat the cause of thesymptoms by: (a) obtaining a biological sample from the subject; (b)obtaining an array of different probes or different sets of probes,wherein a first probe or set of probes selectively interacts with atarget associated with a known cause of the one or more medicalsymptoms, and wherein a second, different probe selectively interactswith a target associated with a therapeutic optimization factor; (c)applying the biological sample to the probes in the array underconditions that enable all of the probes to selectively interact withany targets in the biological sample; (d) detecting interactions; and(e) analyzing interactions to determine a cause of the one or moremedical symptoms and to determine the suitability of a therapeutic agentto treat a cause of the one or more symptoms. In this method, thetherapeutic optimization factor can be tolerance, intolerance, orsusceptibility of the subject or a causative agent to a specific drug,and the target associated with the therapeutic optimization factor canbe a gene in a pathogen that causes susceptibility, resistance, or anidiosyncratic reaction of the pathogen when exposed to a therapeuticagent.

In other embodiments, the invention also features devices. For example,the devices can include (a) a substrate having a surface, wherein thesurface includes a plurality of protrusions having top surfaces; and (b)an array of probes or sets of probes, wherein each probe or set ofprobes selectively interacts with a unique target, and is attached tothe top surface of one of the protrusions. The substrate can be silicon,silicon dioxide, glass, polystyrene, gold, metal, metal alloy, zeolyte,polymer, or other organic or inorganic molecule.

The devices can also include (a) a substrate having a surface, whereinthe surface comprises multiple wells, each well comprising a micromixer;(b) a micromotor connected to each micromixer; and (c) an array ofprobes or sets of probes, wherein each probe or set of probes in thearray selectively interacts with a unique target and is attached withinone of the wells. In certain embodiments, the micromixer is a microfanblade, and the micromotor is an electromagnetic, a chemical, or abiological motor.

Another device includes (a) a substrate having a surface, e.g., a planarsurface; (b) an array of probes or sets of probes, wherein each probe orset of probes in the array specifically binds to a unique target; and(c) an set of linkers, wherein the linkers bind the probes to thesurface, and wherein the linkers have different lengths. The linkers canbe molecules of polyethylene glycol.

In another aspect, the invention features a diagnostic system thatincludes a plurality of devices of the invention, wherein each deviceincludes an array of different probes or different sets of probes, andwherein each probe or set of probes selectively interacts with a targetassociated with a different known cause of a medical symptom or a set ofrelated medical symptoms. The invention also includes a method ofdetermining a cause of one or more medical symptoms exhibited by asubject by (a) assessing the subject's symptoms; (b) selecting one ofthe new devices from the diagnostic system; (c) obtaining a biologicalsample from the subject; (d) applying the biological sample to theprobes on the device array under conditions that enable all of theprobes to selectively interact with any targets in the biologicalsample; (e) detecting interactions; and (f) analyzing interactions todetermine a cause of the one or more medical symptoms. This method canfurther include analyzing interactions to determine the suitability of atherapeutic agent to treat a cause of the one or more symptoms.

In these methods, the cause can be a fungal, bacterial, viral, or othermicrobial cause, genetics or another cause or a combination of causes.The cause can also be vascular, infection/inflammation/autoimmune,neoplasm, drugs, iatrogenic, congenital/developmental/inheritied, orenvironmental exposure/endocrine/metabolic. The sample can be blood,cerebrospinal fluid, urine, sweat, buccal or other swab, a cell sample,or a cell culture. The protein analytes can be antibodies, antigens,glycoproteins, or enzymes. The nucleic acid analytes can besingle-stranded or double stranded, DNA or RNA, or a DNA-RNAcomplex/hybrid or a cell. Furthermore, the probes can be attached to thesubstrate using covalent or non-covalent bonds. For example, the probescan be attached to the substrate using amide or thiol bonds.

In the devices, the wells can have micromixers, such as fans, and canfurther include electrical connections, wherein the electricalconnections connect the mixing devices to an energy, e.g., voltage,source. In addition, the micromixers can be biological molecules poweredby micromotors that run on biologic reactions, e.g., based on ATPase,kinesin, kinesin related proteins, myosin, DNA Helicase, DNA Slidingclamps, nucleic acid based rotaxanes and Pseudo-rotaxanes, circulartriplex forming oligonucleotides (CTFO), duplex DNA; as well as chimerasand derivatives of such proteins and nucleic acids. The protrusions orwells can have mixing device(s) powered by electromagnetic radiation orthe piezoelectric effect or other sources of energy.

In other embodiments, the invention features methods for intelligentlyclustering probes in kit design; reagents and kits for use in the newdevices/systems, methods for interpreting the data and results as wellas making a recommendation for diagnosis; methods for immobilizingbiomolecules in such a manner that their activity is largely preserved(e.g., by the use of cross-linked streptavidin and other protein layersfor attaching capture probes, or spotting solutions with reagents thathelp in stabilizing/preserving biological activity of the probes);methods of dispensing probes in one or more replicates and in geometricpatterns that provide a read-out that can be easily converted to aresult by simple visual inspection (e.g., in “X” patterns); methods forimage storage and processing, and manipulating stored signals to form anew image; new systems/devices that will combine the sample collectionmodules with the diagnostic devices into a single combinationdevice/system (that circumvent the need to transfer biological samplesfrom collection tubes to diagnostic devices); new systems/devices inwhich the sample collection module is separate from the diagnosticdevice module and is easily attached together at any stage, withouthaving to take the sample manually out of the collection module; methodsof preserving the developed slides (e.g., by keeping them sealed inaqueous buffer, e.g., containing BSA, milk proteins, glycerol, trehaloseor other such reagents that preserve the activity of attached probes;methods for performing automated processing of slides (includingscreening, scanning and delivery of results); methods of selecting andusing either single unique probes for each analyte or multiple uniqueprobes for each analyte; and methods for placing optical positioningmarkers for automated image processing and read-out (e.g., that caninclude a row of dilution series for dynamic range determination andinternal calibration on the biochip/microarray).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. For example, definitions ofcommon terms in molecular biology can be found in Benjamin Lewin, GenesVII, published by Oxford University Press, 2000 (ISBN 0-19-899276-X);Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, publishedby Blackwell Science Ltd., 1994 (ISBN o-632-02 182-9); Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive Desk 10Reference, published by VCH Publishers, inc., 1995 (ISBN 1-56081-569-8);and Harrison's Principles of Internal Medicine, 12th Ed., published byMcGraw-Hill, New York, U.S.A., 1991.

A symptom is a measurable and/or observable indication of a disease ordisorder. A symptom can be exhibited by any subject, such as a humanpatient, any animal (e.g., a bird, such as poultry, or a mammal such asa domestic animal such as a dog, cat, cow, pig, or horse), or even aplant that is diseased.

A probe is an antibody, antigen, protein, nucleic acid such as RNA orDNA, or other molecule or compound that interacts with (e.g.,specifically binds to or causes a measurable reaction with) a target oranalyte. A target or analyte is a marker for a disease, diseaseetiology, disorder, treatment, etc., and is thus associated with apossible cause for the one or more symptoms of a specific disease ordisorder. For example, a target can be an antigen expressed by amicrobe, such as a bacterium, virus, fungus, or even a pathogenic plantsuch as algae. A target can also be a biological or chemical moleculeproduced by a microbe, such as an enzyme, a toxin, or a nucleic acid(such as DNA or RNA), or even a small organic molecule, or an inorganiccompound, such as a liquid or gas. A target can also be a specificgenetic sequence indicative of a genetic disorder of the subject beingtested. For example, a genetic disorder can be marked by a mutation of agene, a single nucleotide polymorphism (SNP), an extra copy of a normalchromosome or gene, or a missing gene. A target can also be a marker fora therapeutic optimization factor, such as a microbial gene thatprovides resistance, tolerance, or susceptibility to a particular drug.Such a therapy optimization factor can also be a genetic feature of thesubject that makes the subject resistant, tolerant, or intolerant (e.g.,allergic) to a particular drug.

In each case, the target is detected and/or quantitated by a probe. Theprobe binds specifically to the target, which is associated with one ormore symptoms of a specific disease or disorder. Thus, an interaction ofthe probe with a target, e.g., binding to form a complex, indicates thepresence of a specific cause for the one or more symptoms.

Genetic analysis is the detection or measurement of any target oranalyte that has a genetic basis or cause, e.g., a single nucleotidepolymorphism in a nucleic acid sequence, or the presence or absence of aspecific nucleic acid sequence. Genetic analysis also includes themeasurement, either qualitative or quantitative, of any endogenousphysiologic analyte, such as an mRNA for a specific protein, a specificprotein, or a metabolic product. In addition, genetic analysis includesdetecting a specific epitope of an antibody or an antigen.

A “genetic cause” is any cause that has a nucleic acid basis. Forexample, a genetic cause can be a single nucleotide polymorphism in anucleic acid sequence. It can also be the presence or absence of anucleic acid sequence. A genetic cause can be determined by the presenceof a specific epitope of an antibody or an antigen, or a specificconformation of a protein.

Clinically Intelligent Design is a method of clustering a set or sets ofprobes for analyzing targets based on which targets would be detectedfor a given symptom. This method differs from clustering probes simplybased on the compatibility of different tests in a single assay. Rather,only those tests that pertain to the analysis of one or more specificsymptoms are clustered together. Thus, the method incorporates clinicalintelligence in designing and selecting the probes that are clustered ina single assay.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

The invention provides several advantages. Physicians and/or cliniciansreceive a large spectrum of information rapidly by using the newdevices, and laboratories benefit because the new diagnostic kitsrequire only small amounts of reagents and samples. All relevant testsfor a given symptom can be rapidly analyzed with one device, under thesame reaction conditions, thereby reducing test costs, while providing acomprehensive, standardized result in a rapid fashion. Subjects to betested, such as human patients, benefit significantly because they canreceive a better and quicker diagnosis from their physicians compared tothe situation in which patients are subject to a battery of testsrequiring multiple samples.

The new devices/systems also decrease the risk to health care workers bysimplifying assay procedures, reducing sample size, and decreasing theamount of handling of donor samples required (by reducing the totalnumber of separate/individual tests required in a screening procedure),and thereby reduce the risk of infection.

In addition, the new methods and devices provide facile and low costalterations and augmentations of the devices to include additionaltests, which is cheaper than the laborious and costly process of addingnew tests to a battery of tests conducted separately, each using aseparate sample, e.g., from an individual subject.

Another advantage is that it provides a method for detecting almost anybiological analyte, such as nucleic acids as well as non-nucleic acidcomponents, in a mixture simultaneously. The new devices can also beused for parallel processing of a large number of (same or different)samples, providing a high-throughput environment. For example, multiplesets of microarrays can be deposited onto a single biochip, whichenables screening of multiple patient samples.

The new systems also provide easy and simple read-out of results bysimple visual inspection, and in some embodiments simplify samplehandling by combining sample collection and analysis modules tocircumvent the need to transfer biological samples from collection tubesto diagnostic devices. The new methods and devices can also providebetter and newer sample mixing during an assay, which improves thequality as well as reduces the time needed to perform an assay.

Another advantage is that the microarray-based diagnostic methods can beeasily automated and the new devices can be used with the robots andtechnologies currently used in most clinical testing laboratories. Thiswill cut down on the costs for incorporating this new technology into anexisting laboratory. In addition, the microarray-based diagnosticmethods can be carried out with portable devices.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A is a schematic drawing of symptom-specific, clinicallyintelligent, diagnostic biochip device.

FIG. 1B is a schematic drawing of symptom-specific, diagnostic biochipdevice.

FIGS. 2A to 2C are schematic drawings of three symptom-specific,diagnostic biochip devices, each having a different configuration ofprobes in “arrays of arrays” format. For example, FIG. 2A illustratesprobes in an “X” configuration, FIG. 2B shows probes in a “V”configuration, and FIG. 2C shows probes in a “+” configuration.

FIGS. 3A to 3C are schematic drawings of symptom-specific, diagnosticbiochip devices using a 16-microwell format. FIG. 3A is a top view of anenlarged microwell and a solid support, FIG. 3B is a cross-sectionalview, and FIG. 3C is a view of one micro-well in three dimensions.

FIGS. 4A and 4B are schematic drawings of a new probe attachmenttechnology using molecular linkers, such as polyethylene glycol (PEG),to attach the probes to a solid support. This new attachment technologycan be used in conjunction with the new symptom-specific, diagnosticbiochip devices.

FIGS. 5A to 5C are schematic drawings of another novel attachmenttechnology using a three-dimensional covalently linked mesh ofstreptavidin. FIG. 5A shows a solid support with protective material,but no probes. FIG. 5 B shows a solid support with probes and protectivematerial without cross-linking (a dis-ordered array). FIG. 5C shows asolid support with protective material that is cross-linked into anordered array.

FIGS. 6A to 6C are schematic drawings of the streptavidin attachmenttechnology showing the use of a protective layer (6A) and molecularlinkers that have different lengths, and the differences betweencross-linking (6C) and not cross-linking the linkers (6B).

FIG. 7A is a schematic drawing of a mixing system incorporated into amultiwell biochip device.

FIG. 7B is an enlarged view of a single well in the multiwell biochip ofFIG. 7A.

FIGS. 8A and 8B are an alternative version of the mixing system of FIGS.7A and 7B.

FIGS. 9A to 9C are a schematic drawings of a new inverted array systemof multiwell device, that can be used with symptom-specific, diagnosticbiochip devices. The substrate includes raised or elevated structures,such as cylinders, onto which specific probe arrays, or arrays ofarrays, can be deposited. FIG. 9A shows an enlarged well of themultiwell device shown in FIG. 9B. FIG. 9B shows a cross-sectional view,and FIG. 9C shows three-dimensional views of the wells in the multiwelldevice.

FIGS. 10A and 10B are a schematic drawings of pairs of new invertedarray systems of multiwell symptom-specific, diagnostic biochip devicesin the upright and inverted states, respectively.

FIG. 10C is a diagram of an elevated structure array in which eachstructure includes an embedded capillary, electrical wire, or opticalfiber to provide an electrical or optical readout.

FIGS. 11A to 11E are a series of schematic diagrams of an alternativeinverted array system (top view, FIG. 11A, cross-sectional view, FIG.11B) as used with a microtiter plate (top view FIG. 11C, cross-sectionalview, FIG. 11D). FIG. 11E shows the inverted array inserted into themicrotiter plate.

FIGS. 12A to 12D are a series of schematic drawings of the invertedarray and microtiter plate system of FIG. 11.

FIGS. 13A to 13C are schematic drawings of inverted array devices. Eachelevated structure can have a number of probes attached in an array oran array of arrays format. FIG. 13A shows an array of 96 elevatedstructures in a device. FIG. 13B shows an array of 16 elevatedstructures in a device. FIG. 13C shows a square-shaped elevatedstructure, with 30 such structures in a device. The device can also haveedge-features that help with the correct alignment of these devices withmicrowell plates or with use of these devices in an automatomatedfashion.

FIGS. 14A to 14D are schematic drawings of inverted array devices. FIG.14A shows an array of 96 elevated structures in a device. Each elevatedstructure can have a number of probes attached in an array or an arrayof arrays format. The surface of the elevated structure can also a havethree-dimensional nature. In cross-section, the array of FIG. 14A canhave an elevated sub-structure (14B), a planar sub-structure (14C) or adepressed/dimpled sub-structure (14D) such that one probe is attached toeach of these sub-structures or features.

FIGS. 15A to C and FIGS. 16A to C are schematic representations of athree-dimensional porous array. Such a three-dimensional porous arrayscan be manufactured in a number of ways and these figures illustrate onemethodology in which the three-dimensional solid-substrate is an arrayfilled with holes. The holes are filled with a gel-like matrix or othermaterials such as nitrocellulose membranes. The probes can either bepre-bound to the matrix or can be placed subsequent to matrix depositionstep. FIG. 17 is a schematic diagram of a three-dimensional porous arrayplaced inside of a cartridge-type device.

FIGS. 18A and B and 19A to E are diagrams of another three-dimensionalporous array. Such a three-dimensional porous array can be manufacturedin a number of ways and these figures illustrate one method.

FIGS. 20A to C are schematic diagrams of another three-dimensionalporous array based inverted array device. Each elevated structure can bebased on the 3-D porous array format.

FIGS. 21A to E are representations of a point-of-care deviceimplementation of the 3-D porous array format. This is just one exampleof how this format can be used in point of care biochip devices.

FIGS. 22A and B are schematic drawings of two different microfluidicconcentrator biochips.

FIGS. 23A and B are schematic drawings of two different microfluidicbiochips that are a microarray and microchannel combination chip formultiplexed analyses.

FIGS. 24A to D through FIG. 29 are schematic drawings of a variety ofATPase-based fluid micromixers.

FIGS. 30A to C through FIGS. 33A to C are a series of schematic drawingsof additional biological molecule-based fluid micromixers.

FIGS. 34A to E are a series of representations showing howstrand-invader molecules can achieve hybridization enhancement.

FIGS. 35A to G and FIGS. 36A to H are a series of schematic drawings ofnovel hybridization chambers and their various parts.

FIGS. 37A to C are a series of representations of the principal behindthe new UniScreen Technology, which allows detection of any analyte,such as DNA, RNA, and proteins, in a single multiplexed assay.

FIGS. 38A to D are representations of a three-dimensional porous arraydevice with probes attached to the porous substrate.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

There is a need for a comprehensive diagnostic/screening assay wheretests are clustered in clinically useful formats that enable thephysician and/or a clinician to distinguish and discriminate betweendifferent etiologies based on a specific symptom or set of symptoms. Inthe new diagnostic devices or kits, tests are run in parallel, to avoiddelays in disease diagnosis due to iterative and sequentially performedindividual tests. The new devices can immensely simplify thedifferential diagnosis process.

General Methodology

The present invention provides methods for intelligently combining manytests into one test kit or device. The methods enable the clustering ormultiplexing of tests (e.g., probes) specific to symptoms in aclinically intelligent manner to provide devices and assays forperforming multiple tests in parallel for one or more specific clinicalsymptoms. The new devices include only those probes that can help toconfirm or exclude a particular cause for an observed symptom (e.g., tohelp make an accurate diagnosis).

The format of the new multiplexed devices provides a new approach todiagnostic testing. The new devices detect analytes at the molecularlevel within a biological sample (such as, e.g., blood andblood-derivatives, cerebrospinal fluid (CSF), serum, urine or otherbodily fluids, cell swabs, e.g., from the gums or inner cheek, and cellcultures) using an array having a plurality of probes to which thesample substance is applied. In one embodiment, the medical diagnosticdevice can employ microarray technology to cluster many probes onto asingle biochip. However, the new devices and methods are not limited tobiochips or microarrays. Other technologies can be used to create suchdevices. For example, a multiplexed device can be based on bead arraytechnologies or microfluidic array technologies (from companies such asLuminex, Illumina, Aclara, and Caliper). As described herein, there area number of ways of making multiplexed arrays.

The new methods enable the use of multiple probes that are all bound toa substrate using methods and conditions that keep the immobilized probemolecules biologically active. The multiplexed diagnostic devices alsoenable the simultaneous use of numerous disparate tests/probes under thesame reaction conditions with high sensitivity and specificity.

Another feature of the new methods and devices is that numerous types ofanalytes, including nucleic acids and non-nucleic acid analytes can besimultaneously detected and/or quantitated on the same device. A numberof naturally occurring or synthetic molecules recognize nucleic acidsunder physiological conditions. Examples of such molecules includepolypeptide-based or polyamide probes such as transcription factors(e.g., such as zinc-finger proteins (ZFPs)), Helix-Turn-Helix motifproteins (e.g., GATA-1), immunoglobulin motif proteins (e.g., NFkB,NFAT), and polyamides, such as oligomeric heterocyclic minor groovebinders (MGBs). Advantages of using polyamide molecules (such as ZFPsand MGBs) include: 1) they bind to double-stranded nucleic acids, and 2)they do so under almost the same conditions as proteins used to bind toother proteins and other molecules.

ZFPs are transcription factors in eukaryotes (e.g., in yeasts, plants,and mammals), which contain the Cis2His2 class of zinc finger domains,identified first in the DNA and RNA binding transcription factor TFIIIA,as their DNA-binding modules. This class of zinc finger motifs is uniquein that their DNA binding specificities are highly adaptable; unlikemost other DNA-binding domains, dozens of zinc finger domainscharacterized thus far bind to highly diverse DNA sequences, with eachzinc finger domain able to recognize distinctive DNA binding sites. Eachzinc finger module comprises only ˜30 amino acids and folds into a[beta][beta][alpha] motif, stabilized by chelation of zinc between apair of cysteines from the [beta]-sheet and a pair of histidines fromthe [alpha]-helix. The small globular structure often functions innucleic acid binding and, particularly, in sequence-specific recognitionof DNA, which is the key to function of transcription factors.

DNA binding zinc fingers related to those of the mouse transcriptionfactor Zif268 employ such a simple mode of DNA recognition that theyhave become a useful paradigm in the understanding of protein-DNAinteractions and have been used successfully as scaffolds in the designof DNA binding proteins with predetermined sequence specificity. Thesmall size of the zinc finger limits individual modules to therecognition of only a few adjacent base pairs in duplex DNA, but allowsmultiple tandem modules to wind around the major groove of DNA, thusrecognizing a longer run of bases. In the crystal structure of Zif268fingers bound to DNA, three modules occupy the major groove of the DNAin series, each making base-specific contacts and typically overlappingthree to four basepair subsites. Specificity arises from 1:1interactions between residues of each zinc finger [alpha]-helix and thecorresponding DNA bases. Zinc fingers have also been used to bind toDNA-RNA hybrids, RNA duplexes, and nucleic acids containing modifiedbases.

Simple covalent tandem repeats of the zinc finger domain allow for therecognition of longer asymmetric sequences of nucleic acids. Suchadaptability of zinc finger domains in DNA/RNA recognition can be usedto isolate or design novel proteins with altered DNA/RNA bindingspecificities, and to construct tailor-made nucleic acid bindingproteins that specifically recognize almost any predetermined DNA/RNAsequence. For example, phage display technology can be used to createnovel zinc finger proteins that bind diverse sequences with highaffinity and specificity. Such novel or “designer” zinc finger proteinswith desired nucleic acid binding specificities can serve as efficientprobes for detecting nucleic acid sequences in a sample.

Similarly, MGB polyamides are a class of small synthetic molecules thatbind in a sequence-specific manner in the minor groove ofdouble-stranded DNA with extraordinary affinity and specificity. MGBsuse a chemical recognition code that can distinguish each of the fourWatson-Crick base pairs in the minor groove of DNA. Chemists haveapplied this binding code to design and synthesize a number of differentsuch molecules that specifically recognize a given target sequence inthe human genome. MGBs also bind their target nucleic acids underphysiological conditions. Such novel or “designer” minor groove bindingligands with desired nucleic acid binding specificities can also be usedas efficient probes for detecting nucleic acid sequences in a sample.

Transcription factors, such as ZFPs and small molecule polyamide ligandsthat recognize nucleic acids, such as MGBs, constitute a novel class ofprobes that recognize and detect nucleic acids under physiologicalconditions. Conditions used for binging such agents to their targetnucleic acid sequences are similar to the ones used for detectingproteins and other non-nucleic acid components. Thus, these agents canbe combined with protein and other biologic detecting agents onto asingle support, such as a chip, for a unified screening device (e.g.,UniScreen™). Such a device can detect DNA, RNA, proteins, glycoproteins,polysaccharides, other antigens simultaneously, on a single device (suchas a biochip), and under the same conditions (See FIGS. 37A to C).

The specific biochemical environments required for binding of probemolecules currently limit multi-analyte biochip assays. For instance,proteins necessitate a stable pH and temperature to remain folded andretain optimal binding affinity for the target molecule. Conversely,DNA, PNA, and RNA require thermal cycling for hybridization ofcomplementary strands to occur. This temperature variation would lowerthe binding capability of proteins and in most cases completely denaturethem. Therefore, the possibility of protein and nucleic acid probesbinding target molecules in the same assay has been difficult before thedevelopment of the new methods described herein.

However, there are a number of naturally occurring and syntheticmolecules that recognize nucleic acids by processes such as strandinvasion. Strand invasion involves of a modified nucleic acid sequencethat hybridizes with duplex DNA and is capable of removing a length ofnucleic acid via free energy advantage. Single-stranded DNA, RNA, andpeptide nucleic acid (PNA) molecules accomplish strand invasion underspecific conditions. Chimeras of such molecules also result in astrand-invaded complex. In addition, Epoch Biosciences has created aclass of synthetic probes called selective binding complementaryoligonucleotides (SBCs) that consist of modified nucleotide bases thatform hybrids with target duplex nucleic acids. A feature of SBCs is thatthe two strands do not form a stable duplex with each other, yet theyform a very stable complex with the two strands of a target DNA. Thesemolecules are usually used together, that is, the two complementarysequences are used to perform strand-invasion of DNA.

Hybridization of DNA or RNA targets to DNA/RNA probes attached to abiochip requires that the target be in single-stranded form. We havedeveloped a methodology where a strand-invader, such as PNA, circularDNA/RNA, or one of two SBCs (an illustration is shown in FIG. 34B), canhelp separate the target duplex into a complex with single strandedtarget region. As shown in FIGS. 34A to E, this single-stranded targetcan easily bind to probes bound to a biochip without requiring a thermaldenaturation step on the chip. A second, much smaller, oligo nucleotidecan also be used in the mixture to drive the reaction to completion.

This method is relevant to biochip assays because a DNA probe missing anoligo-size portion of the duplex has extremely high binding affinity forany complementary nucleic acid sequence even under physiologicalconditions. (See, e.g., SBC Oligos, Epoch Biosciences, U.S. Pat. No.5,912,340) and Zhang et al., Nucleic Acids Research, 28, 3332-38, 2000).

Although the new multiplexed diagnostic devices and kits have not beenpreviously described, techniques for attaching individual probes tosolid substrates are described in various publications such as, forexample, U.S. Pat. No. 6,110,426; U.S. Pat. No. 5,763,158; U.S. Pat. No.6,171,797; WO 00540046; U.S. Pat. No. 5,858,804; U.S. Pat. No.5,252,743; U.S. Pat. No. 5,981,180; U.S. Pat. No. 6,083,763; WO 0004390;WO 00104389; WO 00104382; and other related publications.

The new devices/systems include many useful and advantageous features.For example, they can detect analytes under uniform temperature andpressure conditions, and can also have reactive sites/arrays that areeither open or are enclosed in a chamber. These chambers can beflow-through or non flow-through. The devices/systems can also beentirely sealed once a sample has been introduced. In other embodiments,the new systems/devices combine sample collection modules withdiagnostic devices into a unique combined module, which circumvents theneed to transfer biological samples from collection tubes etc. to thediagnostic devices. Alternatively, the systems/devices include samplecollection modules that are separate from diagnostic device modules andthat can easily be connected at any stage, without having to take thesample out of the collection module.

The new multiplexed diagnostic systems enable a number of disparatetests to be performed simultaneously and under the same conditions, withlow cross-reactivity and with high sensitivity and specificity. Thedevices/systems also provide a better dynamic range than currentlyavailable systems, and provide simple data interpretation as well asaccurate diagnostic recommendations.

The new devices/systems also reduce the amount of biological sampleneeded. Individual tests as currently performed each require a certainamount of biological sample. As the number of tests goes up, therequired amount of biological sample also goes up. The new multiplexedassay devices overcome such limitations in testing and samplerequirements, because the sample size stays generally the same, evenwhen new test probes are added to the system.

The new devices/systems also can be used with an “array of arrays”format, which provides a single device that can be used to process alarge number of (same or different) samples in parallel, thus providinga high-throughput environment. For example, by introducing multiple setsof microarrays on a single biochip, one can screen multiple patientsamples with clinically clustered tests in one step.

The new devices can be processed and analyzed using automatedprocessing, such as robotic and computer-controlled screening, scanning,and delivery of results. The new devices, such as diagnostic biochips,can be processed using multiple dyes/colors. Assays can be performedeither simultaneously or sequentially. Assays can use either singleunique probes for each analyte or multiple unique probes for eachanalyte. Different types of assays, such as sandwich immunoassays,competition immunoassays, catalytic antibodies, hybridization, andsingle base extension, can be used in the new methods.

The new methods and systems also provide for image storage andprocessing, and manipulating stored signals to form a new image. Suchmethods can provide test results in formats that are easy to read andinterpret. The new methods also include placing optical positioningmarkers for automated image processing and read-out. For example, such amethod can include a row of dilution series for dynamic rangedetermination and internal calibration on each biochip/microarray. Thenew methods generally provide excellent assay results by improving aswell as optimizing currently used sample mixing conditions during theassay.

In some embodiments, the new methods include methods of delivering theresults via communications networks such as the Internet, telephonesystems, or wireless communication systems. Patients can be given a testID number as well as a second unique identifier (such as a password) atthe time of sample collection. When the results are ready, the patientcan access the communications network (e.g., log in at a web site) andobtain comments from the attending physician as well as a clickablebutton to either download or erase the data after a specifiedtime-period.

Methods of Preparing Clincally Intelligent, Symptom-Specific DiagnosticDevices

The diagnostic devices are based on a variety of substrate-basedtechnologies, such as solid plates, chips, or slides, as well as solidbeads or microparticles. For example, the new devices can use microarraytechnology (see FIGS. 1A and 1B as an example). Glass or siliconmicroscope slides/chips can be used to prepare the devices (FIG. 1 a).Alternatively, a larger membrane can be used to prepare up to 96wells/sites per slide (FIG. 1 b). A number of other materials, such asplastics, polymers, metals, and metal alloys can also be used assubstrates. The device can have one or more sites per slides. FIGS. 13Ato 13C, for example, show schematics of 96-site and 16-site invertedarray device. All the glass slides can be coated with an organic orinorganic material to improve the surface properties as well ascovalently attach the probes to the glass slides. If membranes are usedas the substrate for the probes, they should not require pretreatment,but can be pretreated. A number of different targets are detected in asingle assay by using one or more arrays of immobilized capture probeson the substrate (e.g., slide) surface. Methods of selecting andclustering the probes are described below. Such methods are used todetermine which set of capture probes will be immobilized in aparticular array. Coated glass slides can be purchased from commercialsources or can be prepared using standard techniques. The probes arethen attached to the coated substrate using a variety of techniques.Standard binding techniques can be used, as well as novel probeattachment methods described below.

Selecting Targets For Specific Symptoms

For any given clinical symptom, there can be one, two, dozens, orpossibly hundreds of causative agents or targets for the probes of thediagnostic device. A target can be one or more microbes such bacteria,viruses, mycoplasma, rickettsia, chlamydia, protozoa, plant cells (suchas algae and pollens), or fungi. A target can also be a genetic disordersuch as a single nucleotide polymorphism (SNP), a specific gene that isnot normally present or expressed, or not present in multiple copies, ora mutation in a normally present gene. A target can also be atherapeutic optimization factor. For example, a target can be a specificmicrobial gene that renders a particular microbe susceptible orresistant to a particular drug. A target can also be a particulargenetic sequence in a subject that makes the subject resistant,tolerant, or intolerant (allergic) to a particular drug. These types oftargets can be used to develop a specific, tailored, and optimizedtherapeutic regimen. In addition, the targets can be selected to provideresults that are most accepted by physicians and/or clinicians.

One goal of target selection is to select a number of targets (i.e.,associated with possible causes of a specific symptom) that provides ahigh level of reliability that one of the selected targets is the causeof the symptom, and optionally to select additional targets that can beused to optimize therapy. In other words, the goal is to select targetsthat are the most likely to be the cause of the symptom. For example, ifthere are 50 possible targets that can cause a symptom, but only 10targets are known to cause 90% of the clinically observed instances of agiven symptom, then a diagnostic device might include probes (e.g., 10or more probes) to detect only those 10 targets to provide a sufficientlevel of reliability. This device would not provide a positive result ifthe cause of the symptom in a subject happens to be one of the targetsin the 10% not detected by the device. A more sophisticated diagnosticdevice might include an additional set of probes that are specific for10 more known targets that together with the first 10 targets are knownto cause 99% of the clinically observed instances of the symptom. Eitherdevice can include probes designed to optimize therapy. Of course, otherscenarios are possible.

To provide a high degree of accuracy, several different probes can beused to detect and/or quantitate a single, specific target. For example,one probe can be designed to specifically bind to one epitope of anantibody target, and a second probe can be designed to specifically bindto a second epitope of the same antibody. In another example, one probecan be specific for an enzyme that is produced by a specific microbe, asecond probe can be specific for a specific nucleic acid associated withthat microbe, and a third probe can be specific for and antibody in asubject's bloodstream after exposure to the microbe. In addition,numerous probes of the same type can be clustered into separatelocations or spots on a substrate to ensure that sample is evenlydistributed over the entire array and that even low concentrations oftarget are detected. Two or more probes that recognize differentepitopes of an antibody can also be mixed and placed on the same spot.

In each case, the probes are designed to specifically bind to an analytethat is, or is associated with, a target. For example, if the target isan antibody, the antibody is the analyte. If the target is a microbialgene, then a specific nucleic acid sequence can be the analyte. If thetarget is a genetic disorder in the subject, then the analyte might be aSNP or a specific mutant nucleic acid sequence.

Probe Selection

This section describes the different types of molecules that can be usedas probes on different substrates, such as “chips.” For any giventarget, there can be one or more types of probes that can be used tospecifically bind to the target. For example, if the target is a nucleicacid molecule, e.g., from a subject's or microbe's DNA or RNA, thetarget can be detected using a nucleic acid probe or a protein-based,e.g., polyamide-based probe, such as a zinc-finger binding protein(ZFPs), or a minor groove binder (MGB). If the target is a particularantigen, the probe can be an antibody that specifically binds to thatantigen. If the target is an antibody, the probe can be an anti-idiotypeantibody, or the antigen to which the target antibody is known to bind.

Substrates can be linked to probes that will detect only nucleic acidtargets (NuScreen™ Chip), only non-nucleic acid (e.g., protein-based orpolypeptide-based and other types of targets such as haptens andchemicals) targets (ProScreen™ Chips), or both nucleic acid andprotein-based targets (UniScreen™ Chips).

NuScreen™ Chips: These chips are used for analyzing nucleic acidcomponents of a sample. They can analyze DNA, RNA, or both, and do so intheir single-stranded or double-stranded form. Probes can be XNA based(DNA, RNA, PNA, LNA, HNA, etc.) or protein and polypeptide based(transcription factors, such as ZFPs, and small molecules, such asMGBs), or a combination of both. XNA probes usually bind tosingle-stranded nucleic acids, except for triplex forming oligos thatbind duplexes. Thus, an optional denaturation step can be involved.Preferred probes are based on DNA oligonucleotides 16-40 bases long.ZFPs and MGBs recognize nucleic acids in their double-stranded form andthus require no denaturation step. Target nucleic acids from the samplecan be pre-amplified prior to detection on the NuScreen™ Chip. Thetarget nucleic acids can also be labeled with a detectable moiety duringthe amplification step. Amplification can be done using conventionaltechniques such as PCR, Reverse Transcription—Polymerase Chain Reaction(RT-PCR), in vitro trasnscription (IVT), Nucleic Acid Sequence BasedAmplification (NASDA), Rolling Circle Amplification (RCT) etc.

ProScreen™ Chips: These chips are used for analyzing all othercomponents of a mixture that the NuScreen™ chip cannot detect. Thus,ProScreen™ chips are used to detect, e.g., proteins, polypeptides,peptides, glycoproteins, antigens, haptens, or small organic molecules.Probes can be protein, peptide based or cell-based (such as cellsexpressing specific antibodies), but can also be, for example,glycoproteins, antigens, haptens, small organic molecules, nucleic acidmolecules, and aptamers.

UniScreen™ Chips: These chips are universal screening devices, whichmeans that they can detect almost any kind of target, be it a specificnucleic acid sequence or a protein or something else, with very highspecificity and selectivity. The probes used for detection of allanalytes other than nucleic acids are similar to the ones used inProScreen™ chip. However, protein and peptide-based nucleic acid probescan be used for detecting nucleic acids, such as DNA and RNA, in thesample. An advantage of protein-based probes, such as ZFP and MGBprobes, is that they recognize a specific nucleic acid sequence underphysiological conditions, without any requirements to denature thenucleic acids. Thus, they can be combined with probes that are used todetect targets other than nucleic acids and be effectively used underthe same binding conditions.

This invention utilizes the well-known sequence specific recognitionproperties of certain protein and peptide molecules that bind to nucleicacids selectively. The specific binding reaction does not requiredenaturation of the target nucleic acids and occurs under normalphysiological conditions. Specifically, target nucleic acid molecules donot need to be denatured to a single-stranded form. ZFPs recognize DNA,RNA, and DNA-RNA duplexes. Binding takes place under physiologicalreaction conditions and is specific for each ZFP-nucleic acid sequencepair. Single base changes can easily be probed with this methodology, asthe binding affinity of ZFP is greatly diminished for nucleic acids witheven one base different than the target sequence of the ZFP molecule.This difference in binding is especially pronounced if the differentbase is recognized by the middle finger of a multiple finger (e.g.,three-finger) protein molecule.

Thus, for the first time, the new methods enable the simultaneousdetection of almost any combination of analytes on the same surface andusing the same device, independent of the nature of the analyte. Thedevice that performs such a function is the UniScreen™ Chip. In oneembodiment, biotinylated DNA/RNA target (labeled during PCR/IVT steps)can be used. Labeled nucleic acid targets are captured by ZFPs anddetected using anti-biotin antibodies coupled to streptavidin/HRP. Inaddition, Tyramide Signal Amplification/Rolling Circle AmplificationTechnology (TAS/RCAT) can be used for further signal amplification. Goldon silver staining methods (similar to immunohistochemical stainingtechniques) can also be used.

Substrate Selection and Methods of Attaching Probes

This section describes the different types of substrates (e.g., glassslides) and surfaces that can be used to create diagnostic devices, andprovides examples of different immobilization methods that can be usedto attach probes to these substrates.

In one embodiment, glass slides are used to prepare biochips. Thesubstrates (such as films or membranes) can also be made of silica,silicon, plastic, metal, metal-alloy, anopore, polymeric, and nylon. Thesurfaces of substrates can be treated with a layer of chemicals prior toattaching probes to enhance the binding or to inhibit non-specificbinding during use. For example, glass slides can be coated withself-assembled monolayer (SAM) coatings, such as coatings of asaminoalkyl silanes, or of polymeric materials, such as acrylamide andproteins. A variety of commercially available slides can be used. Someexamples of such slides include 3D-link® (Surmodics), EZ-Rays® (MosaicTechnologies), Fastslides® (Schleicher and Schuell), Superaldehyde®, andSuperamine® (CEL Technologies).

Probes can be attached covalently to the solid surface of the substrate(but non-covalent attachment methods can also be used). In oneembodiment, similar substrate, coating, and attachment chemistries areused for all three—UniScreen™, ProScreen™, NuScreen™-devices. In anotherembodiment, different chemistries are applied.

A number of different chemical surface modifiers can be added tosubstrates to attach the probes to the substrates. Examples of chemicalsurface modifiers include N-hydroxy succinimide (NHS) groups, amines,aldehydes, epoxides, carboxyl groups, hydroxyl goups, hydrazides,hydrophobic groups, membranes, maleimides, biotin, streptavidin, thiolgroups, nickel chelates, photoreactive groups, boron groups, thioesters,cysteines (e.g., for native chemical ligation methods of Muir et al.,Proc. Natl. Acad. Sci. USA, Vol. 95, pp. 6705-6710, June 1998),disulfide groups, alkyl and acyl halide groups, glutathiones, maltoses,azides, phosphates, and phosphines. Glass slides with such chemicallymodified surfaces are commercially available for a number ofmodifications. They can easily be prepared for the rest, using standardmethods (Microarray Biochip Technologies, Mark Schena, Editor, March2000, Biotechniques Books).

In one embodiment, substrate surfaces reactive towards amines are used.An advantage of this reaction is that it is fast, with no toxicby-products. Examples of such surfaces include NHS-esters, aldehyde,epoxide, acyl halide, and thio-ester. Most proteins, peptides,glycopeptides, etc. have free amine groups, which will react with suchsurfaces to link them covalently to these surfaces. Nucleic acid probeswith internal or terminal amine groups can also be synthesized, and arecommercially available (e.g., from IDT or Operon). Thus, nucleic acidscan be bound (e.g., covalently or non-covalently) to surfaces usingsimilar chemistries.

The substrate surfaces need not be reactive towards amines, but manysubstrate surfaces can be easily converted into amine-reactivesubstrates with coatings. Examples of coatings include amine coatings(which can be reacted with bis-NHS cross-linkers and other reagents),thiol coatings (which can be reacted with maleimide-NHS cross-linkers,etc.), gold coatings (which can be reacted with NHS-thiol cross linkers,etc.), streptavidin coatings (which can be reacted with bis-NHScross-linkers, maleimide-NHS cross-linkers, biotin-NHS cross-linkers,etc.), and BSA coatings (which can be reacted with bis-NHScross-linkers, maleimide-NHS cross-linkers, etc.). Alternatively, theprobes, rather than the substrate, can be reacted with specific chemicalmodifiers to make them reactive to the respective surfaces.

A number of other multi-functional cross-linking agents can be used toconvert the chemical reactivity of one kind of surface to another. Thesegroups can be bifunctional, tri-functional, tetra-functional, and so on.They can also be homo-functional or hetero-functional. An example of abi-functional cross-linker is X-Y-Z, where X and Z are two reactivegroups, and Y is a connecting linker. Further, if X and Z are the samegroup, such as NHS-esters, the resulting cross-linker, NHS-Y-NHS, is ahomo-bi-functional cross-linker and would connect an amine surface withan amine-group containing molecule. If X is NHS-ester and Z is amaleimide group, the resulting cross-linker, NHS-Y-maleimide, is ahetero-bi-functional cross-linker and would link an amine surface (or athiol surface) with a thio-group (or amino-group) containing probe.Cross-linkers with a number of different functional groups are widelyavailable. Examples of such functional groups include NHS-esters,thio-esters, alkyl halides, acyl halides (e.g., iodoacetamide), thiols,amines, cysteines, histidines, di-sulfides, maleimide, cis-diols,boronic acid, hydroxamic acid, azides, hydrazines, phosphines,photoreactive groups (e.g., anthraquinone, benzophenone), acrylamide(e.g., acrydite), affinity groups (e.g., biotin, streptavidin, maltose,maltose binding protein, glutathione, glutathione-S-transferase),aldehydes, ketones, carboxylic acids, phosphates, hydrophobic groups(e.g., phenyl, cholesterol), etc. Such cross-linkers can be reacted withthe surface or with the probes or with both, in order to conjugate aprobe to a surface.

Other alternatives include thiol reactive surfaces such as acrydite,maleimide, acyl halide and thio-ester surfaces. Such surfaces cancovalently link proteins, peptides, glycopeptides, etc., via a (usuallypresent) thiol group. Nucleic acid probes containing pendantthiol-groups can also be easily synthesized.

Alternatively, one can modify glass surfaces with molecules such aspolyethylene glycol (PEG). A novel approach to creating such modifiedsurfaces is to use PEGs of mixed lengths (see, e.g., FIGS. 4A and 4B and6A to 6C). Exposed ends of PEGs can be activated with bifunctionalcross-linkers as mentioned above. As shown in FIG. 4B, the variedlengths of PEG linkers create a three-dimensional, rather than a flat,two-dimensional binding environment (FIG. 4A), which provide higherprobe attachment densities because of better packing of the biologicalmolecules upon attachment. Packing of biomolecules, such as proteins,would be higher on a slightly three-dimensional or uneven bindingsurface than on a completely even and flat binding surface.

Yet another alternative is to create a three-dimensional, covalentlylinked mesh of streptavidin or other linker molecule (see FIGS. 5A to6C). For example, piranha-treated glass is first coated with a terminalamine containing silylating agents (e.g.,N-(3-acryloxy-2-hydroxypropyl)-3-aminopropyltriethoxysilane,acryloxytrimethylsilane, triethoxy methyl silane, and aminopropyltriethoxy silane). After a baking step, activated streptavidin (otherproteins such as avidin can also be used) is applied to the surface of ablank slide (activation is done using bifunctional cross-linking groups)(FIG. 5A). This creates a three-dimensional mesh on the glass surface.The streptavidin molecules are linked not only to the glass surface, butare also cross-linked with each other (FIG. 5C) to create an orderedarray. Absent cross-linking, the array is typically dis-ordered (FIG.5B). The cross-linking density can be controlled by the relativeconcentrations of streptavidin and the cross-linkers (NHS-activators,bis-biotin, etc.). After coating with streptavidin, excess NHS-esterscan be quenched with glycine (or other reagents such as ethanolamine,tricine, etc.) or with a layer of BSA, milk-protein, or a number ofother such biochemical reagents to reduce non-specific binding. Theaddition of multi-functional cross-linkers, such as NHS-biotin ormaleimide-biotin, to this surface regenerates active groups ready forcovalently linking (amine- or thio-group containing) probes. Theresulting surface is much more reactive with proteins and other probemolecules (FIG. 5C).

FIGS. 6A to 6C are similar to FIGS. 5A to 5C, but in this example,probes of mixed lengths are used, as also shown in FIG. 4A. The variedlength probes (and/or cross-linkers) provide an uneven bindingenvironment that can provide higher probe attachment densities becauseof better packing of the biological molecules upon attachment.

Many other surface modification alternatives (such asphoto-crosslinkable surfaces and thermally cross-linkable surfaces) areknown to those skilled in the art. Some technologies are commerciallyavailable, such as those from Mosiac Technologies (Waltham, Mass.),Exiqon™ (Vedbaek, Denmark), Schleicher and Schuell (Keene, N.H.),Surmodics™ (St. Paul, Minn.), Xenopore™ (Hawthorne, N.J.), Pamgene(Netherlands), Eppendorf (Germany), Prolinx (Bothell, Wash.), SpectralGenomics (Houston, Tex.), and Combimatrix™ (Bothell, Wash.).

Surfaces other than glass are also suitable for such devices. Forexample, metallic surfaces, such as gold, silicon, copper, titanium, andaluminum, metal oxides, such as silicon oxide, titanium oxide, and ironoxide, and plastics, such as polystyrene, and polyethylene, zeolites,and other materials can also be used. The devices can also be preparedon LED (Light Emitting Diode) and OLED (Organic Light Emitting Diode)surfaces. An array of LEDs or OLEDs can be used at the base of a probearray. An advantage of such systems is that they provide easyoptoelectronic means of result readout. In some cases, the results canbe read-out using a naked eye.

Probes can be deposited onto the substrates, e.g., onto a modifiedsurface, using either contact-mode printing methods using solid pins,quill-pins, ink-jet systems, ring-and-pin systems, etc. (see, e.g., U.S.Pat. Nos. 6,083,763 and 6,110,426) or non-contact printing methods(using piezoelectric, bubble-jet, syringe, electro-kinetic, mechanical,or acoustic methods. Devices to deposit and distribute probes ontosubstrate surfaces are produced by, e.g., Packard Instruments. There aremany other methods known in the art. Preferred devices for depositing,e.g., spotting, probes onto substrates include solid pins or quill pins(Telechem/Biorobotics). Each probe can be deposited in one or morereplicates to achieve better results. Probes can also be deposited insuch a geometric pattern that the read-out can be easily converted to aresult by simple visual inspection (see FIGS. 1-3). For example, probescan be deposited in a square pattern of nine spots (FIG. 1A), an “X”pattern of five spots (e.g., FIGS. 1B and 3A), a “V” or “A” pattern ofthree spots (FIG. 2B), or a “+” of five spots (FIG. 2C).

Fine-Tuning (“Developing”) Devices

After the probes are deposited, the devices/slides/supports aredeveloped using standard techniques according to surface modificationand probe attachment chemistries used. For example, NHS-ester activatedslides, that have amine-group containing probes attached, can bedeveloped by incubated in a humidity chamber (preferably 75%-80%) and at4° Celsius for 2-16 hours. The developed slides, in a preferredembodiment, can be kept sealed in an aqueous buffer until the time oftheir use. The aqueous buffer can also contain Bovine Serum Albumin(BSA), milk proteins, glycerol, trehalose or other such reagents thatpreserve the activity of attached probes. In other embodiments, theslides can be kept in a dry, cool, and dark environment.

Some commonly used blockers are as follows:

1. BSA, e.g., combined with other blockers and surfactants.

2. Casein, a milk-based protein containing indigenous biotin (however,it should be avoided when working with systems involving biotin toprevent interference).

3. Pepticase™ (Casein Enzymatic Hydrolysate): an enzymatic derivative ofcasein.

4. Non-Ionic Surfactants: Tween 20® and Triton X-100® are typical. Whenused in combination with another blocker, a common ratio is 1%Blocker:0.05% Surfactant.

5. “Irrelevant” IgG

6. FSG (Fish Skin Gelatin), pure gelatin or gelatin hydrolysate can alsobe used.

7. Polyethylene Glycol, a versatile blocker, available in a number ofsizes, configurations, and charges can also be used.

8. Sera and non-cross reacting serums, such as horse or fish serum, arevery inert.

9. Polysaccharides and glycoproteins.

10. Commercial Blockers, composites of two or more single blockingsubstances of various molecular weights, can also be used effectivelyover a wide range of conditions.

An alternative blocking methodology is carried out as follows. After theprobes are spotted on the solid surface, the rest of the chip surfacecan be deactivated by irradiating with electromagnetic radiation. Thisstep can be performed after the blocking steps noted above, to denaturethe blocking agents. This step will reduce the antigenic properties ofthe surface agents and will result in lower non-specific binding oftarget molecules during an assay.

Devices

As shown in FIG. 1A, a diagnostic biochip device 20 can contain morethan one array 22, in a multi-reaction site (e.g., multispot ormultiwell) format, such as sixteen reaction sites 24, such asmicrowells, on a solid support 26, such as a slide. In the case of amutiwell device, each microwell 24 can contain probes in an array 22 oran “array of arrays” format. Probes can be deposited in an easy to readgeometrical pattern (here, a square of nine spots). Each microwell isdelimited in that it has partitioned zones. The partitioning can beachieved by chemical treatment or by application of a mask, preferablyhydrophobic, onto the surface of the slide, either prior to, or afterprobe deposition steps. The partitioning can result in the creation ofcylindrical microwells that have a higher sample retaining capacity,compared to supports without wells. Each solid support 26 can includescannable markings 28 (such as a bar code) for computer-controlled,automated processing.

FIG. 1B illustrates a diagnostic biochip device 30 with ninety-sixreaction sites 34, such as microwells on a solid support 36, such as amembraneous slide. Each microwell 34 can contain probes in an array 32or an “array of arrays” format. Probes can be deposited in an easy toread geometrical pattern (here, an “X” of five spots). Solid support 36includes scannable markings 38 for computer-controlled, automatedprocessing. Such markings can be anywhere on the support, including onthe sides or back of the support. These markings allow the support/slideto be read/scanned using conventional scanning devices, such as laserscanners.

FIGS. 2A to 2C illustrate additional probe array configurations. FIG. 2Ashows probes in an “X” configuration. FIG. 2B shows a “V” or “A”configuration, and FIG. 2C shows a “+” configuration.

FIGS. 3A to 3C, show another diagnostic biochip device 40 that containsmore than one array 42, in a multi-reaction site (e.g., multiwell)format, such as sixteen reaction sites 44, such as microwells, on asolid support 46, such as a slide. Each microwell 44 can contain probesin an array 42 or an “array of arrays” format. Probes are deposited in ageometrical pattern (here, an “X” of five spots). Each microwell 44 isdelimited in that it has partitioned zones. Here, partitioning isachieved by applying a mask 43, preferably of a hydrophobic material,onto the surface of the solid support 46, either prior to, or afterprobe deposition steps. The partitioning creates cylindrical microwellsthat have a higher sample retaining capacity, compared to supportswithout wells. The mask 43 can be applied to the support 46 on top of anintermediate organic layer 45 as shown in the cross-section of FIG. 3B.FIG. 3C shows a three-dimensional view of a single microwell 44.

Each solid support 46 can include a scannable ID marking 48 forcomputer-controlled, automated processing. Here there are two sets ofmarkings 48, to allow easier access for scanning, or to providedifferent information on each marking.

One problem with biochip devices is the formation of bubbles duringsample application step. An advantage of the cylindrical shape of thewells is that it has fewer problems with bubbles due to curved walls ofthe microwell. A rectangular shape creates sharp corners, which usuallyretain air-bubbles. A cylindrical shape does not have that problem.

Mixing Apparatus for Use in Microarrays and Biochips

Mixing of samples during incubation is important in microarray assays.Binding efficiency of different target analytes to their respectiveprobes is directly linked to their concentration as well as their rateof diffusion. Mixing a sample during incubation helps increase the rateof diffusion thus giving better and more reproducible results. Thebinding time can also be decreased if efficient means of mixing can beachieved. FIGS. 7A and 7B as well as FIGS. 8A and 8B illustrate solidsupports that include micromixers powered by micromotors (such asmicro-fans and biological motors). These micromotors can be, forexample, electric, magnetic, optoelectronic, or biochemical motors.Mixing can also be achieved by incorporating magnetic beads (such asDynaBeads®) in the sample and using a stirrer underneath the biochip tostir each well.

FIG. 7A shows a solid support 76 with sixteen microwells 74. Eachmicrowell 74 is outfitted with a micromotor 73 including microfan blades75. FIG. 7B shows an enlarged view of a single microwell 74 and one wayof attaching the motor to the slide and depositing probe arrays aroundthis motor in four quadrants. Other probe array configurations arepossible. Electricity is conveyed by wires 77 that run from one or moreelectrical connectors 79, e.g., at one end of slide 76, to eachmicromotor 73. Electricity can also be conveyed by metal or otherconductors deposited onto or into the solid support, e.g., usingstandard printed circuit technology. New miniaturization techniquesallow the entire micromotor to be deposited onto the solid support in asimilar manner. Slide 76 can include computer-readable markings 78 asdescribed above.

FIG. 8A illustrates a solid support 86 with sixteen microwells 84. Eachmicrowell 44 is outfitted with a micromotor 83 including a microfanblade 85. FIG. 8B shows an enlarged view of a single microwell 84 and away of depositing probe arrays around this motor in a circle.Electricity is conveyed by wires 87 that run from one or more electricalconnectors 89, e.g., at one end of slide 86, to each micromotor 83. Asabove, electricity can also be conveyed by metal or other conductorsdeposited onto or into the solid support, e.g., using standard printedcircuit technology. New miniaturization techniques allow the entiremicromotor to be deposited onto the solid support in a similar manner.Slide 86 can include computer-readable markings 88 as described above.

In all of these embodiments, electric motors can be powered usingelectromagnetic sources such as electronic and photonic means. Somemotors can have optically activatable switches that can be switched onusing light. Molecules that can be induced to rotate between twogeometric shapes, such as cis- and trans-stilbene that undergo stereoconversion from one to another configuration upon electromagneticexcitation, are one example of such motors. Biological motors, such asATP synthase, can be powered by electromagnetic sources or by biologicalreactions. For example, ATP synthase can be attached to a bead (forexample, see “Biological machines: from mills to molecules, NatureReviews Molecular Cell Biology 1; 149-152, 2000), and its motor can beattached to another bead. The two beads will rotate with respect to eachother, in the presence of molecules such as ATP, and in the process, mixthe fluids.

A number of biological molecule based fluid micromixers are presented.For example, these micromixers can be based on ATPase (see, e.g., Soonget al.; Science, 290 (5496):1555-1560, 2000; Wang et al., Nature,396:279-282, 1998; Montemagno et al., Nanoscale Biological Engineeringand Transport Group, Cornell University, Nanotechnology, 10:225-231,1999; (http://falcon.aben.cornell.edu.). Micromixers can also be basedon kinesin, kinesin Related Proteins, myosin, DNA Helicase, and DNASliding clamps (see, e.g., Bertram et al., Journal of BiologicalChemistry, 275(37):28413-28420, 2000; O'Donnell et al., Journal ofBiological Chemistry, 270(22):13358-13365, 1995; Hingorani et al., TheEMBO Journal, 18(18):5131-5144, 1999); nucleic acid based rotaxanes andPseudo-rotaxanes (Ryan et al., Chemistry and Biology, 1998); circulartriplex forming oligonucleotide (CTFO) and duplex DNA (Rehman et al.,1999); as well as chimeras and derivatives of such proteins and nucleicacids. FIGS. 24A to 33C illustrate how these can be manufactured andused.

An innovative molecular switch can also be designed for thesemicromixers. Novel synthetic analogs of nucleotide triphosphates, inconjunction with aminoacid modification in the binding site of theprotein micromixers can be used for this purpose. For example,structure-based amino acid changes, as utilized in the studies ofvarious kinases by Shokat and co-workers, can be applied to theseproteins (See, Bishop et al., UNNATURAL LIGANDS FOR ENGINEERED PROTEINS:New Tools for Chemical Genetics, Annu. Rev. Biophys. Biomol. Struct.,29:577-606, 2000). Thus, operations of these micromixers cn be tightlycontrolled using chemical means.

Some of these mixing devices can also be incorporated in the surfacescovering biochips, such as cover slips or hybridization chambers. Anadvantage of such a methodology would be that the mixing apparatus wouldbe compatible with the currently available chip platforms.

Hybridization Chambers

One of the critical issues with current biochip assays is the highvariability in results. One way of overcoming this issue is byperforming the same assay on more than one array or on more than onebiochip. This improves the confidence level of the output. A shortcomingof this approach is that there can still be small variations in theassay conditions, which may affect the results. A better approach wouldbe to perform the assay in such a way that the reaction conditions areidentical. The new systems can include a novel hybridization chamberthat can perform assays on two chips simultaneously and under samereaction conditions. Two biochips are laid one on top of the other, withthe reactive arrays facing each other and separated along the edges witha thin separator. The space left in the middle accommodates the sample,which contacts both of the slides simultaneously. The two slides and theseparator may be enclosed in a chamber. Alternatively, a chamber withpreformed side protrusions can be designed and two biochips can beinserted to make a reaction chamber. FIGS. 35A to E and FIGS. 36A to Eillustrate some embodiments and all of the components of novelhybridization chambers. The interior chamber of these hybridizationmodules can be filled in a variety of ways, such as by using a pipetman,syringe, or needle.

Inverted Array Devices

New inverted array microarray devices are illustrated in FIGS. 9A to12D. These new devices consist of one or more elevated structures orcolumns on a solid support or platform. A device can have one or moresuch structures and the structures can be of any geometric shape andform. The structures can also be vertically straight, angled, ortwisted. The structures can be in the form of one or more arrays.Multiple probes are bound in an array (or an array of arrays) to thesurface of the elevated structures. Thus, each elevated structure,denotes a (multiplexed) reaction site. The device can be used to performreactions simultaneously or sequentially.

Any of the known substrates and chemistries can be used to create such adevice. For example, glass, silica, silicon wafers, plastic, metals andmetal alloys can all be used as the solid support. Elevated structurescan be manufactured by a number of techniques known in the art, such asetching, machining, photolithography, and other microfabricationtechniques. Similarly, probes can be attached to the surface of thesedevices using a number of different methods as described herein.

FIGS. 9A to 9C show one example of such an inverted array device 90.Device 90 can contain more than one array 92, in a multi-reaction site(e.g., multiwell) format, such as sixteen reaction sites 94, such aselevated circular structures, on a solid support 96, such as a slide.Each elevated structure 94 can contain probes in an array 92 or an“array of arrays” format. Probes can be deposited in an easy to readgeometrical pattern (here, a square of nine spots). Each elevatedstructure is delimited in that it has partitioned zones. Thepartitioning is achieved here by application of a mask (or coating) 93,of a hydrophobic or hydrophilic material, onto the surface of slide 96,either prior to, or after probe deposition steps. The partitioning canresult in the creation of cylindrical elevated support structures thathave a higher sample retaining capacity, compared to supports withoutsuch structures. Each solid support 96 can include scannable markings 98(such as a bar code) for computer-controlled, automated processing.

FIG. 9B shows device 90 from a cross-sectional side view. FIG. 9C showstwo different configurations (cubic and cylindrical) of the elevatedstructure 94 on solid support 96 in three dimensions.

FIG. 10A shows two types of inverted array devices 100 in schematicform. Each device includes a solid support 106 and a plurality ofelevated structures 104 (either cubic or cylindrical). The solidsupports 106 can include scannable identification markings 108, such asbar codes or circular codes. FIG. 10B shows the same devices wheninverted for use.

FIG. 10C illustrates an embodiment in which each of the elevatedstructures 104 includes an embedded capillary fiber optic or electrical“tube” 105, which can simplify the assay and the read-out process. Eachof these “tubes” 105 can have one or a set of related probes attached.Each tube, or set of tubes is connected (e.g., either electrically oroptically) with wires or optic fibers 107 that carry a signal from thetubes through or along the solid support 106 and out of the device forresult read-out.

For some applications, the inverted array devices are surrounded by aliquid barrier or wall, to contain sample fluids introduced onto thesurface of the device. In other embodiments, assays are performed usingthe new inverted array devices by incubating the entire device on orwithin a chamber, such as a microtiter plate. In these cases, no sampledelimiting structure or flow barrier is needed on the array deviceitself, because the sample is not placed or poured directly on thedevice, but is held in a separate microtiter plate, to which the deviceis applied. In one embodiment, the device can be lowered into a reactionvessel, such as microtiter plates, to perform assays.

FIGS. 11A to 11E illustrate how an inverted array device 110 is invertedand inserted into a microwell or microtiter plate 111. FIG. 11A shows atop view of inverted array device 110 with 96 elevated structures 114 ona solid support 116 having scannable markings 118. FIG. 11B shows a sideview of device 110. FIG. 11C shows a top view of a microtiter plate 111with microwells 113. In use, the inverted array device 110 is invertedand placed onto the microtiter plate 111 to insert each elevatedstructure 114 into an individual microwell 113. Each microwell containsone sample, and each microwell can contain the same or a differentsample. Sets of 2, 3, 4, 5, 10, or more microwells can also contain thesame sample.

FIGS. 13A to 13C illustrate another embodiment of the inverted arraydevices. The devices can have edge features that help with the alignmentor positioning of these devices with the microwell plates. These edgefeatures can also help with the use of these devices by automatedinstruments.

FIG. 14A presents yet another embodiment of the inverted array devices.Each elevated structure can have a number of probes attached in an arrayor an array of arrays format. The surface of the elevated structure canalso have a three-dimensional configuration. The cross-sectionpossibilities are shown in FIGS. 14B, 14C, and 14D. The sub-structurecan either be elevated (14B), planar (14C), or depressed/dimpled (14D)such that one probe is attached to each of these sub-structures orfeatures.

In other embodiments, the device can form a sealed chip with an enclosedreaction chamber (with holes for sample input, etc.). Assay read-out canbe performed using standard techniques, such as optical methods (e.g.,colorimetry and fluorescence), electrical detection, scanometricdetection, surface plasmon resonance, impedence, capacitance, andchemical sensing (e.g., measuring changes in redox potential).

There are many advantages of this type of device/apparatus. Thisdevice/system is easy to automate, especially with the current roboticsystems. The inverted array system/device can easily be moved from onereaction vessel to another. For example, biological samples can beloaded into one tray and the wash solution into another. The invertedarray can be incubated with the sample and then simply moved to the washplate for washing. Thus, assays can easily be automated and theautomation can be done even on known instruments, which are adept athandling trays and devices that are similar to the microtiter plateformat. Each device can optionally include a “handle” to help move thedevice. A robotic arm can move the device with the help of suctiondevices or with grabbers using standard devices and techniques if nohandle is provided. The inverted array devices also have better mixingcapabilities during assay procedures, because the microtiter plates canbe mechanically moved or stirred in the presence of the “inverted array”device, thus providing mechanical mixing without dislodging the invertedarray device.

In addition, multiple assays can be performed on a single device,because the elevated structures are widely separated from each other. Adevice with multiple elevated structures (an inverted array) can beincubated in a tray with multiple microwells, to perform simultaneousanalyses of multiple samples, or for simultaneous analyses of the samesample with multiple sets of probes and/or under different conditions,or both.

Three-Dimensional Porous Array Devices

Microarray based biochips provide an ideal environment for multiplexedor parallel assays. However, a disadvantage of the system is thatreactions are slow on microarrays due to slow diffusion of analytes. Forexample, in a binding assay, such as antigen-antibody binding, thereaction at a specific spot/site is dependent upon the transportation ofselected molecules to that site and the reaction between probes at thatsite with the analytes (or targets) (see, e.g., Arenkov et. al;Analytical Biochemistry, 278, 123-131, 2000; Timofeev et. al; NucleicAcids Research, 24 (16), 3142-3148, 1996; Van Beuningen R., VicePresident Pamgene International: A Flow Through Porous SubstrateMicro-Array for Post-Genomic Applications). Typically, diffusion oflarge target biomolecules is the slow and limiting factor in the bindingassays, including assays on microarrays. This speed of this process canbe substantially increased by mixing the analytes on the surface of thearray with mechanical, electrical, electronic, optical, optoelectronicor other means. We have devised novel three-dimensional porous arrays toaddress these issues.

New three-dimensional porous microarray devices are illustrated in FIGS.15A to 21E and 38A to 38D. These new devices consist of one or moreporous gel-bound probes in an array or an array of arrays format. Adevice can have one or more such structures and the structures can be ofany geometric shape and form. The structures can also be verticallystraight, angled, or twisted. Thus, each device denotes a (multiplexed)reaction site. The device can be used to perform reactionssimultaneously or sequentially. Any of the known substrates andchemistries can be used to create such a device. For example, glass,silica, silicon wafers, plastic, metals; and metal alloys can all beused as the solid support (see. e.g., Stillman B A, Tonkinson J L,Scleicher and Schuell; Biotechniques, 29(3), 630-635, 2000; Rehmna et.al; Mosaic Technologies Inc., Nucleic Acids Research, 27(2), 649-655,1999).

The device can be manufactured in a number of ways. In oneimplementation, small holes are manufactured in a solidthree-dimensional object using photolithography, etching, drilling, orother techniques known in the art. The holes can be of any geometricshape and can also have slits or grooves. Probes can be immobilized inthese holes using a variety of methods including embedding them in apolymeric matrix. The probes can be separately mixed with apre-polymeric gel and poured into or dispensed or deposited into each ofthe different holes to create polymeric, porous plugs. Subsequently, theprobe-gel mixture can be polymerized using photo-initiators or othermethods known in the art (see, e.g., Arenkov et. al; AnalyticalBiochemistry, 278, 123-131, 2000; Timofeev et. al; Nucleic AcidsResearch, 24 (16), 3142-3148, 1996; Mirzabekov et al.; Methods inMolecular Biology, 170, 17-38, 2001; and Mirzabekov et al.; U.S. Pat.No. 5,981,734, Nov. 9, 1999). The polymerized probe-gel material can besecured in the three-dimensional substrate by application of a secondarymask or a membrane on either or both sides. The securing material canalso have slits or holes. Dumb-bell shaped polymeric plugs can also beused to immobilize probes, especially when the holes have grooves on theoutside. This way the plugs cannot slip or fall out. Such holes andplugs can further be sealed in from two sides with another membrane withor without slits.

There are a number of advantages of this type of device including:

(i) The probes are bound in a three-dimensional porous material.Three-dimensional probe spots result in a higher amount andconcentration of the probe bound to a spot, compared to atwo-dimensional spot (i.e., binding of probes to a flat surface). Thiscorresponds to increased spot resolution of the scanned microarray.

(ii) Porosity of the spot results in a porous array of spots. This givesbetter binding performance, due to enhanced diffusion of the targetmaterial through the entire spot as well as around the array of spots.There is no solid, impenetrable boundary between the porous material andthe fluids on either of the two sides of the porous spot/plug. Thus, thebiological sample being tested on such an array does not encounter anyimpermeable surface while passing through the pad. All the pad-basedbiochips known in the art have a solid-substrate covering any gel-pads(Such as products from Schleicher and Shuell, Motorola, and MosaicTechnologies)(see, e.g., Stillman B A, Tonkinson J L, Scleicher andSchuell; Biotechniques, 29(3), 630-635, 2000; Rehmna et. al; MosaicTechnologies Inc., Nucleic Acids Research, 27(2), 649-655, 1999) thatresult in poor diffusion of biological samples through the pads/spots.

(iii) The diffusion properties of the new devices can be furtherimproved by mechanically mixing the fluid in a direction orthogonal tothe plane of the array. For example, a vacuum suction device or apipetman can be used for this purpose. This can substantially reduce thetime it takes to perform a multiplexed assay. Sample mixing or transportcan also be achieved or increased by using electrophoresis and otherelectronic (e.g., Nanogen, U.S. Pat. No. 6,238,624) or optical means.

(iv) Since the assay time is substantially reduced, this type of devicecan be adapted to manufacture a point-of-care device as well, especiallywhere a single biochip is placed in a container and the fluid mixing ismechanically controlled (See, FIGS. 21A-E).

(v) Post-assay detection of the array results can be simplified andautomated because the position and the size of each probe spot isuniform. This results in lower spot-to-spot and array-to-arrayvariation.

(vi) Almost any material can be used as a substrate, since the problemof background fluorescence of the material is completely eliminated. Theauto-fluorescence of only the probe-gel mixture needs to be considered.

(vii) Almost any attachment chemistry can be used to attach probes tothe porous material. The attachment can be covalent as well asnon-covalent. Thus, this type of format offers a wide variety ofchoices.

(viii) Adopting this type of a design for the microarray devices willsubstantially reduce manufacturing costs.

Additional references of interest include: GeneLogic/HARC, U.S. Pat. No.5,843,767 and http://homer.hsr.ornl.gov/cbps/Genosensors.htm.

Microfluidics Concentrators

New microfluidics-based devices (for example, see P. Chou, M. A. Unger,A. Scherer and S. R. Quake, “Integrated Elastomer Fluidic Lab on aChip—Surface Patterning and DNA diagnostics,” in Proceedings of theSolid State Actuator and Sensor Workshop, Hilton Head, S.C. (2000) andP. Chou, M. A. Unger, A. Scherer and S. R. Quake, “Integrated ElastomerFluidic Lab on a Chip—Surface Patterning and DNA diagnostics,” inProceedings of the Solid State Actuator and Sensor Workshop, HiltonHead, S.C. (2000)) can also be incorporated into the biochip devices orbe used separately. Such microfluidic devices can have chambers orchannels that each contain an array of probes that bind complementaryanalyte targets. This type of an array serves to concentrate relatedanalytes onto a single spot. Bound analytes could be released, usinglabile linkers on probes, and directed into a second channel or chamber.There the analytes could be further analyzed either on a second array ofprobes or in a capillary electrophoretic channel. Thus, each analye isanalyzed in two orthogonal dimensions providing a more accurate result.The microfluidic devices can be made on glass, polymers, plastic,silicon, metals, and a variety of other solid substrates.

As an example, this type of system can be used to perform a proteinprofiling using structural biological principals. There are only alimited number of representative protein folds, such as Immunoglobulindomain, that are used by a majority of proteins in nature. One cangenerate specific probes, such as antibody probes, that recognizespecific protein folds. Such probes can be placed in the central chamberto bind all proteins with a similar fold in a biological sample. Boundproteins can then be further analyzed for specific types of proteins,such as antibodies or cytokines, into the next chamber. Thus, thismicrofluidics system performs biological analyses in two dimensions.

This is a device inside a device type of a system, which utilizes thefast assay time of a microfluidic device for analytical assays. Thereare a number of different ways that this type of device can beimplemented. For example (FIGS. 22A and 22B and 23A and 23B), in onedevice (FIG. 22A), a central chamber has a set of probes, which areplaced at the intersection of an orthogonal artery feeding into anotherchamber. The probes attached at this nodal point are non-specific inthat they bind to a set of target molecules that are unique and yet haveat least one similar characteristic. In a way, these nodes act at pointsin the stream where similar analytes are concentrated. Once this part ofthe assay is complete, the orthogonal artery is activated and transportseach concentrated target set into separate chambers where they arefurther analyzed into unique positions, based on a second set ofinteractions. Thus, this system incorporates two orthogonal detectionprobes and will thus have a much better detection capability. Anotheradvantage of this type of system, besides being a fast and improveddetector, is that it can be combined with other types of biochip devicesfor enhancing their performance.

Some assays require analysis of molecules as well as enzymatic activity.The new methods use a novel microfluidic biochip for such assays. Itwill combine microarrays, for analyses of molecules, with microchannels,for analyses of enzymatic activity and reaction products, on a singlebiochip.

Methods of Using Clinically Intelligent Diagnostic Devices

The new diagnostic devices, e.g., diagnostic kits, are simple to use byphysicians, nurses, clinicians, and/or agricultural worker. Samples fromthe subject (e.g., a human or animal patient, a blood sample from ablood supply, a sample from a plant) are easy to obtain and apply to thediagnostic kits. The results are easily read from a diagnostic kitreader device, e.g., a device that reads fluorescent light or othersignals emitted from the probes of the diagnostic kit.

Specimen collection and purification methods (for subsequent associationto a probe array of the system/device) include all front-end processessuch as biological specimen collection, purification, isolation, andlabeling as required. Most of the protocols are standard protocols andall are published. They are all known to anyone skilled in the art. Asan example, one protocol is described.

Biological specimens, such as fluids (CSF, blood or urine etc.) arecollected in standard collection tubes. In the case of blood or bloodproducts, the serum is separated from nucleated cells using standardprotocols. Serum specimens, CSF, etc. are used for all proceduresdescribed below. Following collection, serum samples should be stored atroom temperature no longer than 8 hours. If the assay cannot becompleted within 8 hours, the sample should be refrigerated at 2-8° C.If the assay cannot be completed within 48 hours, it should be frozen at−20° C. or lower. Frozen specimens should be mixed well after thawingand prior to testing. For NuScreen™ devices, nucleic acids will beisolated from the cells and purified.

Many assay methods are available and are known to anyone skilled in theart. Standard assays will be used in most cases. NuScreen assays will bebased on nucleic acid detection by hybridization. Sample would be put onthe multiplexed test sites and incubated for binding to occur. Singlebase extension (SBE) method, with queried base as the last nucleotide ofthe probe oligo, will be used for polymorphism analyses. Chemical aswell as enzymatic ligation methods, as well as rolling circleamplification, can also be used. Reagents for performing SBE will beadded and the test chamber will be sealed. For SBE (and other) assaysthe SBE reaction will be performed after optimizing (other methods suchas hybridization, ligation, and RCA can also be used). The test samplewill then be washed with a large volume of SSC or other aqueous washingsolutions. This will also remove non-specific binding from the surfaceof the array device, e.g., biochips.

If non-fluorescent nucleotides were used in the SBE reaction, they willbe developed using a secondary molecule labeled with a fluorophore (forexample, a fluorescent streptavidin/antibody, or anHRP-streptavidin/antibody conjugate or an EFL-utilizingmolecule-antibody conjugate or gold-antibody conjugate with subsequentsilver treatment etc.). In a preferred detection method, DNA/RNA will belabeled with biotinylated nucleotides during PCR/IVT. TSA protocol willbe used for detection (from NEN). RCAT (Molecular staging/Amersham) canbe used in place of TSA for signal amplification. Such techniques andreagents are widely known and commercially available. A final washingstep with an aqueous solution will follow to remove unused fluorophoresetc. (detection can be primary or secondary).

ProScreen™ assays will include modified Western blot, ELISA and relatedmethods, primarily for protein, peptides, nucleic acids and otherbiological moieties (competition assays and others can also be used).Samples will be put on the multiplexed test sites and incubated for afew minutes to several hours for binding to occur. Concentration of theprobes on the biochip will be optimized according to the bindingaffinity of various biomolecules to their corresponding probes. Nucleicacid component of the test sample can be amplified and labeled (withfluorophores etc.) separately prior to the biochip assay. Amplified andlabeled nucleic acid fraction can be combined with the non-nucleic acidfraction and then applied to the microarray. Subsequent to the bindingreaction, the test sample will be washed away with a large volume ofphosphate buffered saline (PBS) or another aqueous washing solution.This will also remove non-specific binder from the surface of the chip.

Binding reactions will be developed using secondary molecules labeledwith a reporter group, such as a fluorophore (for example, a fluorescentantibody, or an HRP-antibody conjugate) or an EFL-utilizingmolecule-antibody conjugate or gold-antibody conjugate with subsequentsilver treatment. In one useful method, sandwich ELISA coupled tobiotin/HRP will be used. A TSA step can be used as signal amplificationmethod. RCAT (Molecular staging/Amersham) can be used in place of TSAfor signal amplification. Alternatively, a chemiluminescent orradioactive or electroactive or redox active or IR-active agent etc. canbe used. Such techniques and reagents are widely known and commerciallyavailable. A final washing step with an aqueous solution will follow toremove unused fluorophores etc.

UniScreen™ assays are similar to ProScreen™ assays, but are morecomprehensive and inclusive of a greater variety of target analytes.

The results are monitored by detection and/or imaging of the diagnostickit, such as a biochip for the association(binding/hybridization/extension) of the target molecules/agents onspecific sites in the arrays (within each device) can be achieved byscanning/imaging. Such methods are widely used and the devices toperform these operations are commercially available. There are a numberof commercially available devices that can be used with no or minormodifications. Examples include the GenePix™ system (Axon Instruments,Union City, Calif.), Scanarray (Packard BioSciences, MA), and Arrayworx(Applied Precision, Wash.).

The results are determined by processing the images to determineinformation about the target biological sample such as the presence andamount of specific molecular/other constituents that leads to thescreening output. Software tools will be used to obtain diagnosis fromthe read-out of any test slide. Commercially available softwares such asGenePix Pro (Axon Instruments), Scanarray (Packard), Microsoft® Excel®(Microsoft), and Adobe® Photoshop® (Adobe) can be used, e.g., with minormodifications.

Specific Types of Kits

Human Diagnostic Kits

A wide variety of human diagnostic kits can be created using the methodsand probes described herein. These kits provide information to aclinician or physician about the causes for specific symptoms, orclusters of symptoms, presented by a patient.

Specific examples of human diagnostic kits are in the Examples sectionbelow and include: Headache/fever/meningismus (Meningitis) Kit,Cough/fever/chest discomfort/dyspnea (Pneumonia) Kit, Jaundice (Liverfailure) Kit, Recurrent Infection (Immunodeficiency) Kit, Joint PainKit, and many others.

Human Detection Kits

Kits of this type provide information about the current state of apatient's condition, such as the patient's immunization orimmunocompetance state or the presence of a tropical disease in the body(e.g., a disease not yet showing symptoms), or the condition of amedical product, such as a blood supply or a donated organ.

Specific examples of human detection kits are in the Examples sectionbelow.

Animal Diagnostic and Screening Kits

These kits will allow comprehensive, cost-effective, rapid diagnosis ofnumerous congenital and acquired diseases based on animal's clinicalpresentation of the specific symptoms and/or conditions. In addition,animal exposure to different pathogens or products of pathogens (e.g.,toxins, or the result of immunization) can be evaluated, as well asspecific genes and/or diseases linked to improved breeding (e.g., thesize of the litter, and the meat/milk production). These kits will bespecies-specific. Examples include: Laboratory Mouse Kit, Sheep Kit,Laboratory Rat Kit, Dog Kit, Simian Kit, Racing Horse Kit, Cattle Kit,Chicken Kit, Porcine Kit, Lamb Kit, Fish Kit.

Agriculture Kits

These kits will allow comprehensive, cost-effective, rapid diagnosis ofnumerous congenital and acquired diseases based on plant's clinicalpresentation of the specific symptom. In addition, plant exposure todifferent pathogens will be evaluated, as well as specific genes and/ordiseases linked to improved plant growth (e.g. the size of the plant,the corn/rice production etc). These kits will be species specific. Someof these are listed below: Corn Kit, Cotton Kit, Tobacco Kit, and RiceKit.

Other Kits

The invention covers additional, more specific kits as follows: ForensicKits; Food-borne pathogens (viral and microbial) and antibioticresistance Kit; Inspection of imported goods—agricultural and livestockKit; Pesticide Kit; Inspection of Cosmetics (e.g., Mad Cow Disease) Kit;Bioterrorism Kit (such as smallpox, anthrax, plague, botulism,tularemia, and hazardous chemical agents); and Influenza SurveillanceKit (screens all known strains of influenza).

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 Preparation of Clustered Probe Arrays on Various SolidSupports

A blank 3D-Link™ slide (Surmodics), which contains NHS-ester groups atthe surface of the slides, is used in this example. The slide is placedin a chamber for arraying of probes onto the slide surface. Probemolecules (ZFPs and other polypeptides) are dissolved in a bicarbonatebuffer at pH 8.3, and are spotted onto the glass slides using anarrayer. All the probe molecules contain an amine group for reactionwith the slide surface. The spotting solution can also contain chemicalsthat have low vapor pressure (high boiling point) and that preserve theactivity of probe molecules (such as glycerol, trehalose, andpolyethylene glycol). The spotting is done under controlled conditions(such as humidity, in one instance around 70% relative humidity,temperature, for example around 4° C., pressure, and air-flow).

After the probes are spotted, the slides are put in the developmentchamber for 1-12 hours. The chamber is kept under controlled conditions(such as humidity, temperature, pressure, and air-flow) as well. Theslides are then treated with a blocking buffer (aqueous buffer, pH 8.3containing BSA or other blocking reagents) for an appropriate amount oftime. The slides are then washed and stored.

A blank EZ-Ray® slide (Mosaic Technologies) is treated with an aqueousbuffer containing reducing agents (such as DTT or TCEP) to activate theslide surface into free thiol form. The reducing agents are then washedaway with water and the activated slide can be stored under inertatmosphere in a cool, dark place. Activated thiol-surface is nextconverted to an amine reactive surface. The slide is treated with ahetero-bifunctional cross-linkers, such asN-succinimidyl-3-maleimidopropionate (SMP),N-(11-Maleimido-undecanoyloxy)-sulfo-succinimide (Sulfo-KMUS) (orsimilar agents that contain a thiol-reactive maleimide group on one endand an amine-reactive NHS-ester group at the other), in aqueous bufferand at neutral pH (other reaction conditions, such as a different pH,can also be used). Thiols react with the maleimide moieties of thecross-linker, thus converting thiol-groups into amine-reactive NHS-estergroups. The slide is placed in a chamber for arraying of probes onto theslide surface.

Probe molecules (proteins, such as antibodies, antigens and ZFPs,peptides, such as antigens, haptens and MGBs, glycoproteins,polysaccharides, amine-labeled nucleic acids and other probe molecules)are dissolved in buffers at slightly basic pH (for example, bicarbonatebuffer at pH 8.3) and are spotted onto the glass slides using anarrayer. All the probe molecules contain an amine group for reactionwith the slide surface. Spotting solution can also contain chemicals(such as glycerol, trehalose, and polyethylene glycol) that have lowvapor pressure (high boiling point) and that preserve the activity ofprobe molecules. Spotting is done under controlled conditions (about 70%relative humidity and about 4° C., at normal pressure).

After the probes are spotted, the slides are put into a developmentchamber for 1-12 hours. Conditions in the chamber (humidity,temperature, pressure, air-flow etc.) are controlled as well. The slidesare then treated with a blocking buffer (aqueous buffer, pH 8.3containing BSA) for an appropriate amount of time, washed, and stored.

Xenoslide A™ (aminosilane slides from Xenopore) slides are silanated andready to use as received. They can be stored at room temperature. Asolution of the probes is prepared for spotting. For nucleic acidprobes, the concentration is in the range of 1 ng to 1 ug per ml. Spotsize can be controlled by use of solvent mixtures. Correct choice ofco-solvent will result in lower surface tension of the mixture comparedto water and controlled spreading of the spot. Volatility of the solventmixture and thus the drying time can also be controlled by solventcomposition. Use of a lower volatility co-solvent will increase thedrying time.

DMSO can be used because it is a good solvent for nucleic acids, ismiscible with water in all proportions, and has lower surface tensionand lower volatility than water. Typically, up to 50% DMSO is used.Alternatively, glycerol can be used in place of DMSO. The solution isspotted onto the slide. If using only water, it is helpful to maintain ahumidity of 75-80% for a few minutes to allow binding to take place. DNAcan be cross-linked to the slide by exposing the slide to UV light withup to about 200 millijoules of radiation. The slide is now ready forhybridization.

Xenoslide N™ (nickel chelate surface) slides and nickel chelate coverslips can be used with cobalt chelate surfaces (as per manufacturer).The slides are coated with the chelating agent and are charged witheither nickel or cobalt ions. They are ready to use as received. Theycan be stored at room temperature. A solution of His-tagged probes(e.g., protein, peptide, or nucleic acid) in a neutral pH buffer or aslightly basic buffer is prepared. Tris buffer is a good choice. Thesolution concentration should be in the range of 1-3 ug/ml. The solutionis spotted onto the slide or cover slip, and kept wet for 5-10 minutesby putting into a humid chamber. This allows the binding to take place.The slides are air dried. They are now ready for use in captureexperiments.

Xenoslide D™ (aldehyde surface) slides are chemically modified to have ahigh density of aldehyde groups on the surface. These groups immobilizeamino-labeled DNA, proteins, and peptides by Schiff base chemistry. Asolution of probes (such as amino-labeled oligos) is prepared in aneutral pH buffer at a concentration of 1 to 3 μg/ml. Tris buffersshould not be used because they contain free amine groups that can reactwith the plate surface and prevent oligo binding. The oligos are spottedonto the slide, and the slide is kept wet for 5-10 minutes by insertioninto a humid chamber at a relative humidity of 50-70%. The Schiff basereaction is reversible at acid pH. For greater stability, the Schiffbase can be reduced with sodium borohydride. A 1% solution in water, 30minutes at room temperature is usually adequate. This also blocksunreacted groups. If the reducing step is omitted, the unreactedaldehyde groups should be blocked with a solution of ethanolamine inwater (1% by volume). The slides can be air-dried, and are now ready touse in hybridization tests.

Xenoslide E™ (epoxy surface) slides are chemically modified to have ahigh density of epoxy groups on the surface. The epoxy groups are highlyreactive to primary amino groups and hydroxy groups at high pH. Asolution of the probes, e.g., oligos or polypeptides to be bound, areprepared in a solution at pH 10.5-11 at a concentration of 1-3 μg/ml(proteins can be spotted at a lower pH with a longer incubation time).The probe solution is spotted onto the slide, and the slide is kept wetfor 5-10 minutes by putting it into a humid chamber at relative humidityof 50-70%. Blocking of the slide is usually not necessary because thereaction of the epoxy group at neutral pH is quite slow. Therefore, DNAand oligos will only bind by hybridization. The slide is air-dried andis now ready for hybridization experiments.

Xenoslide S™ (streptavidin surface) slides have a high density ofstreptavidin immobilized on the surface. The binding capacity isapproximately 5 picomoles of biotin binding sites per square cm. Theslides are ready to use as received. Unused slides can be stored intheir container under refrigeration. A solution of biotinylated probes(proteins or oligo at a concentration of 1 to 3 μg/ml) is prepared in aneutral pH buffer such as 1×SSC or phosphate buffer. The probe solutionis spotted onto the slide, and the slide is kept wet for 5-10 minutes byputting it into a humid chamber at a relative humidity of 50-70%. Theslides are air-dried and are then ready for hybridization experiments.

UniScreen™ Biochips can be prepared as follows. Microscope glass slideswith thiols on the surface (such as EZ-Ray™ slides from Mosaic, or Thiolslides from Xenopore) are activated to a free thiol form, and are thenwashed with water and stored under nitrogen atmosphere in a cool, darkplace. The slides are treated with hetero-bifunctional cross-linkers,such as N-succinimidyl-3-maleimidopropionate (SMP),N-(11-Maleimido-undecanoyloxy)-sulfo-succinimide (Sulfo-KMUS) etc.,dissolved in aqueous buffers at neutral pH. The thiols react with themaleimide moieties of the cross-linker, converting them intoamine-reactive compounds. Probe molecules are dissolved in buffers withslightly basic pH (for example, bicarbonate buffer at pH 8.3) and arespotted onto the glass slides using an arrayer. The spotting is doneunder controlled humidity (around 70% relative humidity) and temperature(around 16° C.) conditions.

After the probes are spotted, the slides are put into the developmentchamber for 1-12 hours. The chamber is kept under controlled conditions(such as 70% RH, 16° C.). The slides are then treated with a blockingbuffer (aqueous buffer, pH 8.3 containing BSA and other reagents) for anappropriate amount of time, and are then washed and stored.

Example 2 Jaundice (Liver Failure) Kit

This kit allows comprehensive, cost-effective, rapid diagnosis ofnumerous diseases/conditions based on a patient's clinical presentationof jaundice/liver failure. Diagnosis of genetic, autoimmune, andinfectious diseases is based on the precise detection of specific genemutations or other markers (such as microbial-specific sequences, orautoreactive antibodies) using DNA, RNA, and protein (spotted antigenicproteins and specific mAbs) chips. Jaundice kits are focused on theetiologic considerations of jaundice. In addition, therapeutic markersmay be included to test for different potential therapeutic options.Briefly, one or more of three groups of etiological conditions areevaluated: A) autoimmune hepatitis, B) viral-induced hepatitis, and C)genetic diseases causing jaundice and/or liver enlargement. See, e.g.,Feldman: Sleisenger & Fordtran's Gastrointestinal and Liver Disease, 6thed. (W. B. Saunders Company 1998); McFarlane, “IG: The Relationshipbetween autoimmune markers and different clinical syndromes inautoimmune hepatitis,” Gut 42:599-602, 1998; Manns, M P, “Liver/KidneyMicrosomal Autoantigens,” in Autoantibodies (ed: Peter J B and Y.Shoenfeld, Elsevier, 1996), pp 462-466; and Lee: Wintrobe's ClinicalHematology, 10th ed. (Lippincott Williams & Wilkins, 1999).

The kits may include probes that detect targets for potentialtherapeutics.

The kits will include probes that detect at least five or more of thefollowing targets:

1) Anti-LKM-1 antibodies (IgG and IgM)—the major target antigen of LKM-1antibodies has been identified as cytochrome P450 2D6, a microsomalprotein found in the endoplasmic reticulum.

2) Anti-mitochondrial M2 antibody—M2 antigens can be used as probes. M2antigens have been located in the inner mitochondrial membrane and havebeen found to be part of the pyruvate dehydrogenase complex and havemolecular weights of 50 and 70 kD.

3) Hepatitis A infection—this virus can be detected indirectly bydetecting IgG Anti-HAV antibodies using purified recombinant humanhepatitis A antigens as probes.

4) Hepatitis B infection—purified recombinant human hepatitis B antigens(HBcAg, HBsAg, HbeAg) can be used as probes.

5) Hepatitis C infection—purified recombinant human hepatitis C antigen(NS3, NS4, NS5, and core regions antigens) can be used as probes.

6) Hepatitis D infection—purified recombinant human hepatitis D antigencan be used as probes.

7) Hepatitis E infection—purified recombinant human hepatitis E antigencan be used as probes.

8) CMV infection—CMV immediate-early antigens (pp 65) or DNA inperipheral-blood leukocytes can be used as probes to hasten thediagnosis of CMV disease in certain populations, including organtransplant recipients and persons with AIDS.

In addition, the kits will include probes that detect gene mutationsand/or allelic variations that can cause the elevation ofbilirubin/liver injury. For example, some probes in the jaundice kitswill be designed to detect the following mutations/allelic variations:

Dubin-Johnson Syndrome; Hyperbilirubinemia Type I; Acute HepaticPorphyria; Delta-Aminolevulinate Dehydratase Deficiency; PorphobilinogenSynthase Deficiency; Alagille Syndrome; Arteriohepatic Dysplasia;Cholestasis with Peripheral Pulmonary Stenosis; Alpha-1-AntitrypsinDeficiency; Carbamoylphosphate Synthetase 1Deficiency; CarbamylPhosphatase Deficiency; Carbamyl Phosphate Synthetase Deficiency;Carnitine-Acylcarnitine Translocase Deficiency; Citrullinemia;Ferrochelatase Deficiency; Heme Synthetase Deficiency; Fatty AcidOxidation Disorder, Unspecified; Fructose 1,6 Bisphosphatase Deficiency;Galactosemia; Galactose Epimerase Deficiency; Galactose-1-PhosphateUridyltransferase Deficiency; NGlutaricacidemia Type II;Glutaricaciduria Type II; Glycogen Storage Disease Type I/Ia;Glucose-6-Phosphatase Deficiency; Von Gierke Disease; Glycogen StorageDisease Type III; Cori Disease; Debrancher Deficiency; Forbe Disease;Glycogen Storage Disease Type IV; Brancher Deficiency; Glycogen StorageDisease Type IX; Glycogen Storage Disease Type VIII; PhosphorylaseKinase Deficiency of Liver; Glycogen Storage Disease Type Ib;Glucose-6-Phosphate Translocase Defect; Glycogen Storage Disease TypeVI; HERS Disease; Hereditary Coproporphyria; Coproporphyrinogen OxidaseDeficiency; Harderoporphyria; Hereditary Fructose Intolerance;Fructosemia; Hereditary Hemochromatosis; Long Chain 3-Hydroxyacyl-CoADehydrogenase Deficiency; Acute Fatty Liver; Disease of Pregnancy;HELLP; Hemolysis; Enzymes, and Low Platelets; LCHAD Deficiency;Trifunctional Protein Deficiency; Long Chain Acyl-CoA DehydrogenaseDeficiency; LCAD Deficiency; Medium Chain 3-Ketothiolase Deficiency;MCKAT Deficiency; Medium Chain Acyl-Coenzyme A Dehydrogenase Deficiency;MCAD Deficiency; Mucopolysaccharidosis Type II; Hunter Syndrome; MPS II;Mucopolysaccharidosis Type IIIB; MPS IIIB; Sanfilippo Syndrome Type B;Mucopolysaccharidosis Type IIIC; MPS IIIC; Sanfilippo Syndrome Type C;Mucopolysaccharidosis Type IVB; MPS IVB; Morquio Syndrome Type B;Mucopolysaccharidosis Type VI; Arylsulfatase B Deficiency; MPS VI;Maroteaux; Lamy Syndrome; Mucopolysaccharidosis Type VII; GlucuronidaseDeficiency MPS; MPS VII; Sly Syndrome; Niemann-Pick Disease WithoutSphingomyelinase Deficiency; Niemann-Pick Disease Type C; Niemann-PickDisease Type D; Niemann-Pick Disease, Nova Scotian Type; OrnithineTranscarbamylase Deficiency; OTC Deficiency; Phosphorylase KinaseDeficiency of Liver and Muscle; Polycystic Kidney Disease, Recessive;ARPKD; PKD, Infantile; PKD, Recessive; Salla Disease; Sialic AcidStorage Disease; Sialidosis; Glycoprotein Neuraminidase Deficiency; MLI; ML1; Mucolipidosis; Wilson Disease; Wolman Disease; Cholesterol EsterStorage Disease; and/or Zellweger syndrome; Cerebrohepatorenal Syndrome.

To use a jaundice kit, blood is drawn from the patient. The serum isseparated from the nucleated cells. Serum specimens are used for allprotein-based procedures performed on a polypeptide-based array or chip.Following collection, the serum is separated from the clot. Serumsamples are stored at room temperature no longer than 8 hours. If theassay is not completed within 8 hours, the sample is refrigerated at 2to 8° C. If the assay will not be completed within 48 hours, or forshipment of the sample, the sample should be frozen at −20° C. or lower.Frozen specimens must be mixed well after thawing and prior to testing.Peripheral blood leukocytes (PBL) are isolated by percoll gradient,counted, and frozen at 20° C. or lower. For DNA and RNA chips, the DNAand RNA is purified from these cells.

The following diagram shows how the blood sample is tested:

Protein chip technology: A contact-printing robot is used to create adense array of immobilized proteins on glass slides to create thejaundice kits. Covalent attachment to aldehyde-derivatized glass occursthrough a Schiff's base at several protein surface positions. Excessreactivity is quenched with a layer of phospholipids, which also helpsto reduce nonspecific binding (peptide and small protein arrays will bemade on activated phospholipid monolayers). The proteins/peptidescontain a stable structure. Even after they have been attached to thewells of a polystyrene microwell plate, they continue to preserve theirantigenicity in their native state (e.g., these probes are fullyrecognized by autoreactive/and/or anti-viral antibodies from patientssera). After the blood sample is applied to the surface of the probearray, the sample is washed off, leaving only target analytes bound tothe probes. Thereafter, standard antibodies, e.g., monoclonalantibodies, or IgG or IgM antibodies, attached to labels or reportergroups, such as fluorescent labels (e.g., FITC and rhodamine), areapplied to the array, and bind to any analytes attached to the probes,in the manner of a standard ELISA assay.

As an example, the target analyte LKM-1 antibody is detected usingpurified full-length recombinant human cytochrome P450 2D6 antigen boundto the surface of a glass slide. Diluted patient sera are added,allowing any LKM-1 antibodies present to bind to the immobilizedantigen. Unbound sample is washed away and a FITC (green)-labeledanti-human IgG antibody and a rhodamine (red)-labeled anti-human IgM isadded. After washing away any unbound anti-human mAbs, the intensity ofthe color is measured using standard techniques and instruments. Theassay is evaluated by measuring and comparing the color intensity thatdevelops in the patient samples with the color in a control sample.

M2 antibodies can be detected in a similar manner using purifiedmitochondria M2 antigen (also known as pyruvate dehydrogenase) bound tothe support.

Anti-hepatitis A antibodies can be detected using purified recombinanthuman hepatitis A antigens bound to the surface of a glass slide.Diluted patient sera is added, allowing any Hepatitis A antibodiespresent to bind to the immobilized antigen. Unbound sample is washedaway and a FITC (green)-labeled anti-human IgG antibody and a rhodamine(red)-labeled anti-human IgM is added. After washing away any unboundanti-human mAbs, the intensity of the color is measured. The assay canbe evaluated as described above. Similar methods are used to detectother hepatitis antibodies.

CMV infection can be detected by detecting CMV immediate-early antigens(pp 65) or DNA in peripheral-blood leukocytes can hasten the diagnosisof CMV disease in certain populations, including organ transplantrecipients and persons with AIDS. The detection of CMV DNA incerebrospinal fluid by the polymerase chain reaction is useful in thediagnosis of CMV encephalitis or polyradiculopathy. On the other hand,detection of CMV viremia is a better predictor of acute infection.

Various genetic causes for jaundice are detected using nucleic acidprobes, or protein- or polyamide-based probes, which specifically bindto mutant forms of genes or alleles known to be associated with causesfor jaundice as listed above. These probes are prepared using standardtechniques, and are then spotted onto a support or substrate along withother jaundice symptom-specific probes described herein.

Example 3 Fever/Skin Rash/Weight Loss (Autoimmune) Kit

This kit allows comprehensive, cost-effective, rapid diagnosis ofnumerous diseases/conditions based on a patient's clinical presentationof autoimmunity (systemic autoimmune diseases have frequentlyoverlapping clinical pictures consisting of fever, skin rash, skindiscoloration, and weight loss). Diagnosis of autoimmune diseases isbased on the precise detection of autoreactive antibodies, specific genemutations or other markers (such as autoimmune prone HLAs) using DNA,RNA, live cells, and protein (spotted antigenic proteins and specificmAbs) chips. Briefly, one or more of three groups of etiologicalconditions are evaluated: A) Systemic and organ-specific autoimmunediseases, B) HLAs that are associated with specific autoimmune diseases,C) Detection of gene mutations that result in the autoimmune syndrome,D) Deficiencies of early and late complement components associated withautoimmune diseases, and E) Therapeutic markers to test potentialtherapeutic options. See, e.g., Ruddy: Kelley's Textbook ofRheumatology, 6th ed. (W. B. Saunders Company 2001); Allergy: Principlesand Practice, 5th ed. Middleton et al. (eds), (Mosby-Year Book, 1998);and Lee: Wintrobe's Clinical Hematology, 10th ed. (Lippincott Williams &Wilkins 1999).

The autoimmune disease kits will include probes that detect at leastfive or more of the following targets:

A. Antibodies against the following “self” antigens:

Anticardiolipin-purified cardiolipin antigen is used as the probe; ANA(antinuclear antibodies-antigens); SM; RNP; SS-A; SS-B; Scl-70(DNA-topoisomerase-1); Jo-1 (histidyl-tRNA synthetase); ASCA's mannose(anti-Saccharomyces cerevisiae antibodies); Beta2 glycoprotein(apolipoprotein H); Collagen 3 (IV) collagen chain); Cathepsin G;Cationic protein 57 (CAP-57); Elastase; Histones (H2A-H2B-H3-H4);Gliadin; IgA; IgG; IgM; Lactoferrin; LKM-1 (cytochrome P450 2D6); LKM-2(cytochrome P450 2C9); LKM-3 (uridine diphosphate glucoronosyltransferases) type 2; Mitochondria M2, M5, or M6; Myeloperoxidase (MPO);PART poly-ADP-ribose polymerase; Phosphoproteins (diagnostics of SLA);P0; P1; P2; Ribosome P (carboxyl-terminal 22 amino acid peptide); Serineprotease 3 (PR3); ssDNA; dsDNA; Thyroid M (thyroid microsomal antigen);Thyroid T (thyroglobulin); Thyroid peroxidase (TPO); TM; and/or Tissuetransglutaminase (tTG).

B. HLA and autoimmune diseases:

In many autoimmune diseases, there is association of particular HLAantigens in populations of individuals with certain diseases. Probes aredesigned to detect HLAs such as: HLA B27; HLA B38; HLA DR8; HLA DR5; HLADw4/DR4; HLA Dw3; 7HLA DR3; HLA DR4; HLA B5; HLA Cw6; HLA A26; HLA B51;HLA B8; HLA Dw3; HLA B35; HLA DR2; HLA B12; and HLA A3

C. Detection of gene mutations that result in the autoimmune syndrome,such as: Fas; FasL; and the Canale-Smith syndrome.

D. Deficiencies of early and late complement components associated withautoimmune diseases. This list includes known mutations resulting in alack of function of different components of the complement cascade.These mutations are associated with the autoimmune syndrome: C1 (C1q,C1r, C1s); C4; C2; C1 inhibitor; C3; D; Properdin; I; P; C5, C6, C7, C8,and C9.

These kits may also include the following markers:

E. Therapeutic markers to test potential therapeutic options.

For all of these target analytes, corresponding antigens are known andcan be isolated, purified, and used as probes.

To use an autoimmune kit, blood will be drawn from the patient andtreated as described in Example 2. The following diagram shows how theblood sample is tested:

Systemic and organ-specific autoimmune diseases will be assayed usingprotein, live cells, DNA, or RNA chip technology similar to thatdescribed in Example 2 to detect antibodies and antigens in patientsera. Systemic autoimmunity encompasses autoimmune conditions in whichautoreactivity is not limited to a single organ or organ system. Thisdefinition includes systemic lupus erythematosus (SLE), systemicsclerosis (scleroderma), rheumatoid arthritis (RA), chronicgraft-versus-host disease (GVHD), and the various forms of vasculitis.The inference that a disease is autoimmune is made based on the presenceof autoantibodies and the localization in diseased tissue of antibodyand complement.

Specific antigens and autoantigens (as probes) are spotted onto asupport as described in Example 2. Auto-IgG and IgM auto-antibodiesagainst the target analytes are used to visualize the binding or thetarget analytes to the probes as described herein.

To detect HLA and autoimmune diseases, a protein, live cells, DNA, orRNA chip can be used as described above. All mAbs against differentclass I and class II HLAs associated with autoimmune diseases areavailable. Thus, these mAbs against human HLAs are spotted onto asupport. These mAbs specifically bind to HLA class I and class IIproteins isolated from the surface of nucleated cells (these proteinswill be stripped from the cell surface by enzymatic reaction aspreviously described). The secondary mAbs used for detection will bemAbs anti pan-class I and pan-class II, recognizing all alleles withinthe class.

Example 4 Recurrent Infection (Immunodeficiency) Kit

This kit allows comprehensive, cost-effective, rapid diagnosis ofnumerous diseases/conditions based on a patient's clinical presentationof immunodeficiency/recurrent infections. Children with recurrentinfections are among the most frequent types of patients seen by primarycare physicians. Most patients with recurrent infections do not have anidentifiable immunodeficiency disorder. Evaluations of immune functionshould be initiated for children with clinical manifestations of aspecific immune disorder or with unusual, chronic, or recurrentinfections such as (1) two or more systemic bacterial infections (e.g.,sepsis, osteomyelitis or meningitis), (2) three or more seriousrespiratory or documented bacterial infections (e.g., cellulitis,draining otitis media, or lymphadenitis within 1 year), (3) infectionsoccurring at unusual sites (e.g., the liver or a brain abscess), (4)infections with unusual pathogens (e.g., Aspergillus spp, Serratiamarcescens, Nocardia spp, or Pseudomonas cepacia), and (5) infectionswith common childhood pathogens but of unusual severity.

The new immunodeficiency kit approach to the diagnosis ofimmunodeficiency diseases is based on the precise detection ofinfectious such as anti-viral antibodies and genetic markers such asspecific gene mutations, and/or other markers (such as presence ofimmunoglobulins or complement components). The following etiologicalconditions are evaluated: A) Detection of viruses causingimmunodeficiency, B) detection of immunoglobulin classes, C) Detectionof specific immunoglobulins with specificity against common antigens, D)detection of mutations/allelic variations that result inimmunodeficiency, E) detection of the gene mutations that result incomplement deficiencies, and F) detection of therapeutic markers to testpotential therapeutic options. See, e.g., Bone: Pulmonary & CriticalCare Medicine (Mosby-Year Book, Inc., 1998); Allergy: Principles andPractice, 5th ed. Middleton et al. (eds.) (Mosby-Year Book, 1998); andLee: Wintrobe's Clinical Hematology, 10th ed. (Lippincott Williams &Wilkins 1999).

The immunodeficiency kit will include probes that detect at least fiveof the following targets:

A. Detection of viruses causing immunodeficiency: HIV infection;Epstein-Barr Virus (EBV) infection

B. Detection of immunoglobulin classes: IgA; IgG1; IgG2; IgG3; IgG4;and/or IgM.

C. Detection of specific immunoglobulins with specificity against commonantigens:

Spotted tetanus antigen; Spotted diphteria antigens; Spotted Haemophilusinfluenzae antigens; and/or Spotted pneumococci's antigens.

D. Detection of mutations/allelic variations that result inimmunodeficiency: A) SCID associated with defective cytokinesignaling—gammac; Jak3; IL-2; IL-2Ra; and IL-7Ra; B) SCID associatedwith TCR related defects—CD3g; CD3e; and ZAP70; C) HLA class IIdeficiency—CIITA; RFX5; and RFXB; D) HLA class I deficiency (bareleukocyte syndrome)—TAP1 and TAP2; E) Immunodeficiency associated withdefects in enzymes other than kinases—ADA deficiency and PNP deficiency;F) X-linked hyper-IgM—CD40 ligand; G) X-linked agammaglobulinemia(Bruton)—Btk; H) Non-X-linked agammaglobulinemia—m heavy chain; I)Wiskot-Aldrich Syndrome—WASP; J) Ataxia telangiectasia—ATM; K) DiGeorgeanomaly—21q; L) Autoimmune lymphoproliferative syndrome—Fas; M)XLP—SH2D1A/SAP; N) TRAPS—TNFRSF1A; and/or O) Susceptibility tomicrobacterial infections—IFN-gammaR1; IFN-gammaR2; IL-12p40.

E. Detection of the gene mutations that result in complementdeficiencies: C1 (C1q, C1r, C1s); C4; C2; C1 inhibitor; C3; D;Properdin; I; P; C5, C6, C7, and/or C8.

These kits may also include the following markers.

F. Detection of therapeutic markers to test potential therapeuticoptions

For all of these target analytes, corresponding antigens, antibodies,and/or nucleic acids are known and can be isolated, purified, and usedas probes.

To use an recurrent infection kit, blood will be drawn from the patientand treated as described in Example 2. The following diagram shows howthe blood sample is tested:

Viruses and genetic mutations causing immunodeficiency are assayed usingprotein, live cells, DNA, RNA chip technology similar to that describedin Example 2 to detect antibodies, antigens, genetic mutations, andallelic variations in patient samples using the techniques describedherein.

Example 5 Sore Throat (Pharyngitis) Kit

This kit allows comprehensive, cost-effective, rapid diagnosis based ona patient's clinical presentation of sore throat. This kit testspotential infectious agents including bacteria, viruses and otherpathogens. In addition, this kit will test for different therapeuticsincluding such things as bacterial resistance toward some antibiotics.Diagnosis of specific pathogens causing sore throat/pharyngeal painpresentation is based on the precise detection of specific antigens,specific microbial DNA/and or RNA, and specific microbial DNA conferringantimicrobial resistance toward antibiotics. One or more of thefollowing groups of etiological conditions are evaluated: A) Viraldiseases resulting in sore throat, B) bacterial and other pathogensresulting in sore throat, and C) therapeutic markers to test potentialtherapeutic options. See, e.g., Bone: Pulmonary & Critical Care Medicine(Mosby-Year Book, Inc. 1998).

The kits will include probes that detect five or more of the followingtargets:

A. Viral detection—In most instances, the kits will include familyspecific reagents (where applicable, types and subtypes will bedetected): Rhinovirus; Coronavirus; Adenovirus (types 3, 4, 7, 14);Herpes simplex virus (types 1 and 2); Parainfluenza virus (types 1-4);Influenza virus (types A and B); Coxsackievirus A (types 2, 4-6, 8, 10);Epstein-Barr virus; Cytomegalovirus; and/or HIV-1.

B. Bacterial and other pathogens—In most instances, the kits willinclude family specific reagents (where applicable, types and subtypeswill be detected):

-   -   I. Bacterial detection: Streptococcus pyogenes (group A        beta-hemolytic streptococci); Group C beta-hemolytic        streptococci; Neisseria gonorrhoeae; Corynebacterium        diphtheriae; Corynebacterium ulcerans; Arcanobacterium        haemolyticum (Corynebacterium haemolyticum); Yersinia        enterocolitica; Treponema pallidum; Chlamydia pneumoniae;        Mycoplasma pneumoniae; and/or Mycoplasma hominis (type 1).    -   II. Detection of antibodies against the beta-hemolytic        Lancefield group A Streptococcus: Streptozyme;        Antideoxyribonuclease-B; and/or Antistreptolysin-O.

These kits may also include the following markers:

C. Therapeutic markers to test potential therapeutic options, such as:Beta-lactamase.

For all of these target analytes, corresponding antigens, antibodies,and/or nucleic acids are known and can be isolated, purified, and usedas probes.

To use the new sore throat kits, pharyngeal swab is taken from thepatient's tonsils. Also blood can be drawn. Serum will be separated fromthe nucleated cells. Serum specimens and swab material are used for allprotein, DNA, and RNA based procedures performed on protein, DNA, andRNA biochips that include appropriate probes spotted onto supports.Following collection, the serum and nucleic acids are treated asdescribed in Example 2. The following diagram shows how the blood sampleis tested:

To detect microbial antigens, DNA, RNA, genes, and antibodies, protein,live cells, DNA and RNA biochips as described herein can be used.

Example 6 Cough/Fever/Chest Discomfort/Dyspnea (Pneumonia) Kit

This kit allows comprehensive, cost-effective, rapid diagnosis based ona patient's clinical presentation of lower respiratory tract symptoms.This kit tests both potential infectious (bacteria, viruses and otherpathogens) and genetic components that might result in lower respiratorytract symptoms. In addition, this kit will test for differenttherapeutics including such things as bacterial resistance towardcertain antibiotics. Respiratory tract symptoms are among the mostcommon acute problems seen in office practice; the majority are limitedto the upper airway. The cough, fever, chest discomfort, and dyspneathat can accompany lower respiratory diseases provoke great concern inthe patient. This new kit will contain panels of probes for pathogensknown to cause lower respiratory tract symptoms. Briefly, groups causingthe following etiological conditions will be evaluated: A) bacterialdiseases resulting in Pneumonia/bronchitis, B) viral and othernon-bacterial pathogens resulting in Pneumonia/bronchitis, C) autoimmunedisease resulting in inflammation of lung tissue, (D) Poisons andChemicals resulting in inflammation/irritation/destruction of lungtissue, and (E) Therapeutic markers (such as antiobiotic resistancegenes) to test potential therapeutic options. See, e.g., Bone 1998, andAllergy: Principles and Practice, 5th ed. Middleton et al. (eds)(Mosby-Year Book, 1998).

In particular, pneumonia is an infection of the pulmonary parenchyma.Various bacterial species, mycoplasmas, chlamydiae, rickettsiae,viruses, fungi, and parasites can cause pneumonia. Identification of theetiologic microorganism is of primary importance, since this is the keyto appropriate antimicrobial therapy. However, because of the seriousnature of the infection, antimicrobial therapy generally needs to bestarted immediately, often before conventional laboratory confirmationof the causative agent. The new kit can also be used to detect causativeagents related to biological warfare or terrorism.

These lower respiratory tract symptom kits will contain probes thatdetect five or more of the following targets:

A. Bacteria may include (spotted mAbs against these pathogens or DNA orRNA specific probes). In most instances, family specific reagents willbe used (where applicable, types and subtypes will be detected):Streptococcus pneumoniae; Staphylococcus aureus; Group A streptococci;Haemophilus influenzae; Klebsiella pneumoniae; Proteus mirabilis; E.Coli; Pseudomonas aeruginosa; Moraxella (Branhamella) catarrhalis;Legionella pneumophila; Porphyromonas gingivalis; Prevotellamelaminogenica; Fusobacterium nucleatum; Actinomyces spp.; Spirochetes;Anaerobic streptococci; Fusobacteria; Mycoplasma pneumoniae;Mycobacterium tuberculosis; Bacillus anthracis; Yersinia pestis;Francisells tularensis; Coxiella burnetti (Q fever) and/or Yersiniaenterocolitica.

B. Viral and other non-bacterial pathogens may include—In mostinstances, family specific reagents will be used (where applicable,types and subtypes will be detected) Influenza A and B; Adenoviruses;Respiratory Syncytial Virus; Parainfluenza virus; Cytomegalovirus;Varicella (varicella-zoster virus); Variola major (small pox); Rubeola;Blastomyces spp.; Chlamydia psittaci; Coxiella burnetii; Aspergillus;Noccardia; Candida; Pneumocystis Carinii; Histoplasmosis; and/orCoccidiodomycosis.

C. Detection of Autoimmune diseases resulting in inflammation of lungtissue such as Wegener's Granulomatosis—detection of anti-PR3antibodies.

D. Detection of Chemicals and Poisons resulting ininflammation/irritation/destruction of lung tissue such as:

-   -   I. Poison: such as ricin toxin, and    -   II. Chemical weapons: such as Distilled Mustard (HD), Lewisite        (L), Mustard Gas (H), Nitrogen Mustard (HN-2), Phosgene Oxime        (CX), Hydrogen Cyanide, Chlorine (CL), Diphosgene (DP), Nitrogen        Oxide (NO), Perfluororisobutylene (PHIB), Phosgene (CG), Red        Phosphorous (RP), Sulfur Trioxide-Chlorosulfonic Acid (FS),        Teflon and Perfluororisobutylene (PHIB), Titanium Tetrachloride        (FM), and/or Zinc Oxide (HC).

These kits may also include the following markers:

E. Therapeutic markers (such as antiobiotic resistance genes) to testpotential therapeutic options such as: Beta-lactamase

For all of these target analytes, corresponding antigens, antibodies,and/or nucleic acids are known and can be isolated, purified, and usedas probes.

To use the lower respiratory tract symptom kit, sputum and/or bronchialwashings are taken from patients. Also, blood is drawn. Serum will betreated as described in Example 2. Serum specimens and swab material areused for all protein, DNA and RNA based procedures performed on aprotein, DNA and RNA chip. Viruses and genetic mutations, as well asbacteria and other agents, causing sore throat, are assayed usingprotein, DNA, and/or RNA chip technology described herein to detectantibodies, antigens, genetic mutations, and allelic variations inpatient samples using the techniques described herein.

Example 7 Joint Pain Kit

This kit allows comprehensive, cost-effective, rapid diagnosis based ona patient's clinical presentation of joint pain symptoms. This kit testsboth potential infectious, autoimmune and genetic components that mightresult in joint pain symptoms. In addition, this kit will test fordifferent therapeutics. The kits will include targets in one or more ofthe following etiological groups: A) Systemic and organ-specificautoimmune and infectious diseases resulting in joint pain, B) HLAsassociated with specific joint diseases/pain, and C) genetic mutationresulting in joint diseases/pain, and D) therapeutic markers to testpotential options. See, e.g., Ruddy: Kelley's Textbook of Rheumatology,6th ed. (W. B. Saunders Company 2001).

In particular, joint pain is often caused by musculoskeletal disorders,which generally classified as inflammatory or noninflammatory.Inflammatory disorders can be infectious (infection with Neisseriagonorrhoea or Mycobacterium tuberculosis), crystal-induced (gout,pseudogout), immune-related [rheumatoid arthritis (RA), systemic lupuserythematosus (SLE)], reactive (rheumatic fever, Reiter's syndrome), oridiopathic. Noninflammatory disorders can be related to trauma (rotatorcuff tear), ineffective repair (osteoarthritis), cellular overgrowth(pigmented villonodular synovitis), or pain amplification(fibromyalgia). Many serologic tests for rheumatoid factor, antinuclearantibodies, complement levels Lyme disease antibodies, antistreptolysin0 (ASO) antibodies, or Ig rheumatoid factors are carried out fordetection of these diseases.

These kits will include probes designed to detect five or more of thefollowing targets:

A. Systemic and organ-specific autoimmune diseases resulting in jointpain:

-   -   1. Detection of antibodies against following “self” antigens:        Streptozyme; Antideoxyribonuclease-B; Antistreptolysin-O; human        IgA; human IgG; human IgM; Anticardiolipin; ANA (antinuclear        antibodies-antigens): SM; RNP; SS-A; SS-B; Scl-70        (DNA-topoisomerase-1); Jo-1 (histidyl-tRNA synthetase); ssDNA;        dsDNA; ASCA's mannose (S. cerevisiae); LKM-1; LKM-2; LKM-3;        and/or Mitochondria M2.    -   2. Detection of infection with pathogens that could result with        joint pain: Borellia burgdorferi; Treponema pallidum; Yersinia;        Campylobacter; Salmonella; Shigella; hepatitis A virus;        hepatitis B virus; hepatitis C virus; hepatitis D virus;        hepatitis E virus; Haemophilus influenzae; Staphylococcus        aureus; gram-negative bacteria (this is a gram-family specific        probe); Streptococcoccus pneumoniae; streptococcoccus (family        specific probe); and/or Neisseria gonorheae.

B. HLA associated with joint diseases/joint pain: HLA-B27 and/or DRw52.

C. Genetic mutations resulting in joint diseases: HGPT-gene(“Lesch-Nyhan syndrome or Hypoxanthine-Guanine PhosphoribosyltransferaseDeficiency); Gene C282Y; and/or Gene H63D.

These kits may also include the following markers:

D. Therapeutic markers to test potential therapeutic options.

For all of these target analytes, corresponding antigens, antibodies,and/or nucleic acids are known and can be isolated, purified, and usedas probes.

To use a joint pain kit, blood will be drawn from the patient andtreated as described in Example 2. The following diagram shows how theblood sample is tested:

Viruses and genetic mutations, as well as bacteria and other agentscausing joint pain, are assayed using protein, live cell, DNA, and/orRNA chip technology described herein to detect antibodies, antigens,genetic mutations, and allelic variations in patient samples using thetechniques described herein.

Example 8 Headache/Fever/Meningismus (Meningitis) Kit

This kit allows comprehensive, cost-effective, rapid diagnosis based ona patient's clinical presentation of headache, fever, and meningismus(stiff neck). This kit tests both infectious (viruses, bacteria andother pathogens) and genetic components that might result in the symptompresentation of headache, fever and meningismus. In addition, this kittests for different therapeutics including such things as antibioticresistance. The classic clinical presentation of adults with bacterialmeningitis includes headache, fever, and meningismus, often with signsof cerebral dysfunction. Nausea, vomiting, rigors, profuse sweating,weakness, myalgias, and photophobia are also common. The kits willinclude probes for targets in the following etiological groups: A)Infectious markers: Viral, bacterial and other pathogens causingmeningitis, B) genetic markers: Diagnosis of the deficiencies in theterminal complement cascade (C5-C9, properidin) C) Therapeutic markersto test potential therapeutics. See, e.g., Goetz: Textbook of ClinicalNeurology, 1st ed. (W. B. Saunders Company 1999).

The new headache/fever/meningismus kits will contain probes that detectat least five or more of the following targets:

A. Infectious Markers:

-   -   1. Bacteria and Other Pathogens: Haemophilus influenzae,        Neisseria meningitidis, Streptococcus pneumoniae, Listeria        monocytogenes, Streptococcus agalactiae, Propionibacterium        acnes, Staphylococcus epidermidis, Enterococcus faecalis,        Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa,        Salmonella spp., Nocardia spp., Mycobacterium tuberculosis,        Spirochetes (such as Treponema pallidum (syphilis), Borrelia        Burgdorferi (Lyme diseases, Leptospira spp.), and Rickettsiae        (such as Rickettsia rickettsii (Rocky Mountain spotted fever),        Rickettsia conorii, Rickettsia prowazekii (epidemic or        louse-borne typhus), Rickettsia typhi (endemic or murine        typhus), Rickettsia tsutsugamushi (scrub typhus), Ehrlichia        spp.).    -   2. Viruses: Nonpolio enteroviruses (echovirus 11; echovirus 9;        coxsackievirus B5; echoviruses 30, 4, and 6; coxsackieviruses        B2, B4, B3, and A9; echoviruses 3, 7, 5, and 21; and        coxsackievirus B1, enteroviruses 70 and 71); Mumps virus;        Arboviruses (Flaviviridae, The mosquito-borne California enc.        Virus, St. Louis enc. Virus, Eastern equine enc. Virus, Western        equine enc. Virus, Venezuelan equine encephalitis viruses and        Tick-borne Colorado tick fever); Herpesviruses (Primarily herpes        simplex virus type 2, but also herpes simplex virus type 1,        varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, and        human herpesvirus 6); Lymphocytic choriomeningitis virus; Human        immunodeficiency virus; Adenovirus; Parainfluenza virus types 2        and 3; Influenza virus; Measles virus; and/or Polio virus.

B: Genetic markers: such as the terminal complement components: C5, C6,C7, C8, C9, and Properidin.

These kits may also include the following markers:

C. Therapeutic markers to test potential therapeutics such as:Beta-lactamase.

For all of these target analytes, corresponding antigens, antibodies,and/or nucleic acids are known and can be isolated, purified, and usedas probes.

To use a meningitis kit, cerebrospinal fluid (CSF) and blood will bedrawn from the patient. Blood will be treated as described in Example 2,and CSF will be treated using standard techniques. The following diagramshows how the blood sample is tested:

Viruses and genetic mutations, as well as bacteria and other agents,causing headache/meningitis, are assayed using protein, live cell DNA,and/or RNA chip technology described herein to detect antibodies,antigens, genetic mutations, and allelic variations in patient samplesusing the techniques described herein.

Example 9 Diarrhea Kit

This kit will allow comprehensive, cost-effective, rapid diagnosis basedon a patient's clinical presentation of diarrhea. This kit will testinfectious agents including bacteria, viruses and other pathogens aswell as genetic and autoimmune components that can result in diarrhea.In addition, this kit will test for different therapeutics includingsuch things as bacterial resistance toward some antibiotics. Also, thepresence of chemical agents will be evaluated. Probes are selected andused as described herein to detect known targets associated with thesesymptoms.

The following groups of etiological conditions are evaluated: A)Bacteria resulting in diarrhea, B) viruses and other pathogens resultingin diarrhea, C) genetic factors involved in diarrhea, D) autoimmunediseases resulting in diarrhea, E) chemical agents resulting indiarrhea, F) Therapeutic markers to test potential therapeutic options.

These diarrhea kits will contain probes that detect five or more of thefollowing targets:

A. Bacteria resulting in diarrhea: Bacillus cereus, Staphylococcusaureus, Clostridium perfringens, Vibrio cholerae, enterotoxigenicEscheria coli, Klebsiella pneumoniae, Aeromonas species,Enteropathogenic and enteroadherent E. coli (O157:H7), Giardiaorganisms, Clostridium difficile, Hemorrhagic E. coli, Salmonella,Campylobacter, Aeromonas species, Vibrio parahaemolyticus, Yersinia,Shigella species, enteroinvasive E. coli, Bacillus anthracis,Clostridium botulinum

B. Viruses and other pathogens resulting in diarrhea: Cytomegalovirus,Herpes simplex, Enteropathogenic Adenovirus, Rotovirus (Group A, B, C),Calicivirus, Astrovirus, Cryptosporidium, Septata intestinalis,Microsporidium, Entercytozoon bienusi, Isospora belli, Cyclosporaspecies, Giardia lamblia, Entamoeba histolytica, Leishmania donovani,Blastocystic hominis, Pneumocystis carini, Histoplasma, Coccidioides,Candida albicans, Cryptococcus

C. Genetic diseases involved in diarrhea: Acute Hepatic Porphyria;Delta-Aminolevulinate Dehydratase Deficiency; Porphobilinogen SynthaseDeficiency, Amyloidosis Type I; Amyloid Polyneuropathy, Andrade orPortugese Type; Amyloidosis, Portugese Type; Amyloidosis, Swedish Type,Beckwith-Wiedemann Syndrome, Cystic Fibrosis; CF, Dubin-JohnsonSyndrome; Hyperbilirubinemia Type II, Epidermolysis Bullosa Letalis withPyloric Atresia; Aplasia Cutis Congenita with Gastrointestinal Atresia;Carmi Syndrome, Erythropoietic Protoporphyria; ErythrohepaticProtoporphyria; Ferrochelatase Deficiency; Heme Synthetase DeficiencyEthylmalonic Encephalopathy, Familial Adenomatous Polyposis; APC;Adenomatous Polyposis Coli; FAP; Gardner syndrome, FamilialDysautonomia; Riley-Day Syndrome, Familial Gastric Cancer, FamilialHibernia Fever; Familial Periodic Fever; TRAPS, Familial MediterraneanFever; Recurrent Polyserositis, Hereditary Coproporphyria;Coproporphyrinogen Oxidase Deficiency; Harderoporphyria (Included),Hereditary Non-Polyposis Colon Cancer; HNPCC; Lynch syndrome,Hermansky-Pudlak Syndrome; HPS, Multiple Endocrine Neoplasia Typ; MEN1,Ornithine Transcarbamylase Deficiency; OTC Deficiency, Pearson Syndrome;Sideroblastic Anemia w/Marrow Cell Vacuolization & Exocrine PancreaticDysfxn, Peutz-Jeghers Syndrome; Hamartomatous Intestinal Polyposis; PJS,Phosphoglycerate Kinase Deficiency; PGK Deficiency, PseudoxanthomaElasticum, Dominant; PXE, Dominant Pseudoxanthoma Elasticum, Recessive;PXE, Recessive Pyruvate Kinase Deficiency, Townes-Brocks Syndrome; TBS,Wolman Disease; Cholesterol Ester Storage Disease von Hippel-LindauSyndrome; VHL

D. Autoimmune diseases resulting in diarrhea: Antibodies against thefollowing “self” antigens that are associated with autoimmune diseasescausing diarrhea, such as

ASCA's mannose (anti-Saccharomyces cerevisiae antibodies);

These kits may also include the following markers:

E: Chemical agents resulting in diarrhea such as: Adamsite (DM),Diphenylchloroarsine (DA), Diphenylcyanoarsine (DC)

F. Therapeutic markers to test potential therapeutic options such as:Beta-lactamase.

Example 10 Vaginal Discharge and/orBleeding/Abdominal/Pain/Nausea/Vomiting/Temperature (Vaginitis/PID) Kit

This kit will allow comprehensive, cost-effective, rapid diagnosis basedon a patient's clinical presentation of vaginitis/pelvic pain. This kitwill test potential infectious agents including bacteria, viruses andother pathogens. In addition, this kit will test for differenttherapeutics including such things as bacterial resistance toward someantibiotics. Diagnosis of specific pathogens causing sorethroat/pharyngeal pain presentation is based on the precise detection ofspecific antigens, specific microbial DNA/and or RNA, and specificmicrobial DNA conferring antimicrobial resistance toward antibiotics.One or more of the following groups of etiological conditions areevaluated: A) Viral diseases resulting in vaginitis/pelvic pain, B)bacterial and other pathogens resulting in vaginitis/pelvic pain, C)therapeutic markers to test potential therapeutic options.

The kits will include probes that detect five or more of the followingtargets:

A) Viral detection—In most instances, the kits will include familyspecific reagents (where applicable, types and subtypes will bedetected): Human papilloma virus (HPV); Molluscum contagiosum; Herpessimplex virus (HSV) type 1 and 2; Human immunodeficiency virus (HIV);Hairy leukoplakia (Epstein-Barr virus)

B). Bacterial and other pathogens—In most instances, the kits willinclude family specific reagents (where applicable, types and subtypeswill be detected): Treponema pallidum; Chlamydia trachomatis; N.gonorrhoeae; Escherichia coli; Bacteroides species; anaerobic cocci;Calymmatobacterium granulomatis; H. ducreyi; Mycoplasma hominis;Ureaplasma urealyticum; C. trachomatis; Candida albicans

These kits may also include the following markers:

C). Therapeutic markers to test potential therapeutic options such as:Beta-lactamase.

Example 11 Skin Discoloration/Pain/Ulcer (Skin) Kit

This kit will allow comprehensive, cost-effective, rapid diagnosis basedon a patient's clinical presentation of skin rash. This kit will testinfectious agents including bacteria, viruses and other pathogens aswell as genetic and autoimmune components that can result in skin rash.In addition, this kit will test for different therapeutics includingsuch things as bacterial resistance toward some antibiotics. Also, thepresence of chemical agents will be evaluated. Probes are selected andused as described herein to detect known targets associated with thesesymptoms.

The following groups of etiological conditions are evaluated: A)Bacteria resulting in skin rash, B) viruses and other pathogensresulting in skin rash, C) genetic factors involved in skin rash, D)autoimmune diseases resulting in skin rash, E) chemical agents resultingin skin rash, F) Therapeutic markers to test potential therapeuticoptions.

A. Bacteria resulting in skin rash: In most instances, the kits willinclude family specific reagents (where applicable, types and subtypeswill be detected): Staphylococcus aureus; Group A streptococci; Anthrax,Treponema pallidum, Chlamydia trachomatis, N. gonorrhoeae, Escherichiacoli, Bacteroides species, Anaerobic cocci, Calymmatobacteriumgranulomatis, H. ducreyi., C. trachomatis, Candida albicans, Yersiniapestis, Tinea, Candidiasis (moniliasis), Tinea versicolor, Pityrosporumfolliculitis.

B. Viruses and other pathogens resulting in skin rash: In mostinstances, the kits will include family specific reagents (whereapplicable, types and subtypes will be detected): Human papilloma virus(HPV), Molluscum contagiosum, Herpes simplex virus (HSV) type 1 and 2,Hairy leukoplakia (Epstein-Barr virus), variola major (smallpox),arenaviruses, filoviruses, Bunyaviruses, and flaviviruses

C. Genetic diseases involved in skin rash: Hermansky-Pudlak Syndrome*;

Lactate Dehydrogenase Deficiency*; LDH Deficiency

Pseudoxanthoma Elasticum, Recessive*; PXE, Recessive

Peutz-Jeghers Syndrome*; Hamartomatous Intestinal Polyposis; PJS

Pachyonychia Congenita*; Jackson-Lawler Syndrome; Jadassohn-Lewandowsky

Oculocutaneous Albinism Type 1 (Tyrosinase Related)*; OCA1;Oculocutaneous

Pseudoxanthoma Elasticum, Dominant*; PXE, Dominant

Neurofibromatosis Type I*; NF1; Von Recklinghausen Disease

Neurofibromatosis Type II*; NF2

D. Autoimmune diseases resulting in skin rash:

Antibodies against the following “self” antigens such as:Anticardiolipin-purified cardiolipin antigen is used as the probe; ANA(antinuclear antibodies-antigens); SM; RNP; SS-A; SS-B; Scl-70(DNA-topoisomerase-1); Jo-1 (histidyl-tRNA synthetase); Beta2glycoprotein (apolipoprotein H); Collagen 3 (IV) collagen chain)Elastase; Histones (H2A-H2B-H3-H4); Gliadin; IgA; IgG; IgM; Lactoferrin;PART poly-ADP-ribose polymerase; Phosphoproteins (diagnostics of SLA);P0; P1; P2; Ribosome P (carboxyl-terminal 22 amino acid peptide);

These kits may also include the following markers:

E: Chemical agents resulting in skin rash such as: Distilled Mustard(HD), Lewisite (L), Mustard Gas (H), Nitrogen Mustard (HN-2), PhosgeneOxime (CX), Phenodichloroarsine (PD), Sesqui Mustard

F. Therapeutic markers to test potential therapeutic options such as:Beta-lactamase.

Example 12 Immunization/Immunocompetence Kits

Immunization represents a remarkably successful and very cost-effectivemeans of preventing infectious diseases. Because of routine childhoodimmunizations, the occurrence of once common contagious diseasesdeclined markedly in the United States and other countries in the secondhalf of the 20th century. Public health programs based on vaccinationhave led to global eradication of smallpox, elimination of poliomyelitisfrom the Americas and possibly from the world in the near future, andgreater than 95% reduction in the United States and other countries ofinvasive Haemophilus influenzae type b (Hib) disease. In the UnitedStates, immunization has almost eliminated congenital rubella syndrome,tetanus, and diphtheria and has reduced the incidence of rubella andmeasles to record low rates.

Infants and children in this country routinely receive vaccines against10 diseases: diphtheria, tetanus, pertussis, poliomyelitis, measles,mumps, rubella, Hib infection, hepatitis B, and varicella. Rotavirusvaccine is also recommended, with the realization that universalimmunization may require additional time and resources. Hepatitis Avaccine is recommended for some groups of children. More than 50immunobiologic products are licensed in the United States. Despite thisremarkable success, many people are not adequately immunized. Reasonsfor this result include, inter alia, (1) lack of appropriateimmunization in childhood, (2) low quality of administered vaccines, and(3) immunoincompetence of the host at the time of vaccination.

The new devices and methods can be used to evaluate both theimmunocompetence status of patients, and the immune response towardsvarious pathogens. In addition, other parameters of the immune systemcan be evaluated. Kits for such analyses will contain probes for one ormore of: a) antibodies against viral and bacterial pathogens that areadministered with vaccine; b) viral and bacterial antigens that areadministrated as vaccine; and c) probes for genes related with recurrentinfection (see Recurrent infection kit). The probes are selected andused as described herein to detect known targets associated with thesesymptoms.

Example 13 Blood Assaying Kits (BloodBank/Transfusion) Kits

Clinically intelligent bloodbank screening diagnostic kits aremanufactured using the new methods described herein. These kits allowcomprehensive, cost-effective, rapid diagnosis/screening of numerousdiseases/genetics characterization that preclude blood transfusion/organdonation.

The list of pathogens/genetic markers that need to be tested is strictlyregulated/required by the Food and Drug Administration (FDA). The newkits include all tests required and/or recommended by the FDA and by theAmerican Association of Blood Banks. In addition, due to theircost-effectiveness, the kits can include other assays recommended orunder investigation for pathogens and genetic markers. Currentrequirements by the FDA are that each unit of blood must be tested afterblood is drawn. The tests include assays for: ABO group (blood type), Rhtype (positive or negative), and any unexpected red blood cellantibodies that can cause problems in the recipient. Screening tests arealso performed for evidence of donor infection with hepatitis viruses Band C, human immunodeficiency viruses (HIV) 1 and 2, humanT-lymphotropic viruses (HTLV) I and II and syphilis. BloodBank kit willcontain panels of all scientifically accepted screening tests fordiagnosis of different infectious diseases (HIV, Syphilis, HBV, HCV,CMV), genetic characteristics (HLA, Rh antigens) that could preventand/or influence blood transfusion/organ transplantation. Kits for theseblood analyses contain probes that detect one or more of the followingtargets: (1) viral and bacterial pathogens that excludes blood/organtransplantation, (2) Characterization of HLAs, blood groups and Rh (andother related) blood groups. All blood tested positive is discarded.

The kits include probes designed to detect five or more of the followingtargets that are analyzed using either UniScreen, ProScreen, and/orNuScreen chips:

I. Viruses: hepatitis A virus; hepatitis B virus; hepatitis C virus; HIV1 and 2; Human T-Lymphotropic Virus, Types I and II (Anti-HTLV-I, —II);Treponema pallidum; Borrelia burgdorferi; CMV; Malaria; Epstein-Barrvirus (EBV); Babesiosis; and/or Chagas' Disease;

II. Blood groups, Rh types or HLA in donor sera: HLA Typing; ABO BloodGroup System; Rh System (Rh d, Rh e, and Rh c); Other blood groups (Kell(K), Duffy (Fy), Kidd (jK), MN, P, Lewis (Le), Lutheran (Lu), Velsystem, and/or Wright (Wra).

For all of these target analytes, corresponding antigens, antibodies,and/or nucleic acids are known and can be isolated, purified, and usedas probes.

To use the new blood screening kits, donated blood will be collected.Serum will be separated from the nucleated cells. Serum specimens willbe used for all protein-based procedures performed on a protein chip.Following collection, the serum should be separated from the clot andtreated as described in Example 2.

The following diagram shows how the blood sample is tested:

Viral and bacterial pathogens that exclude blood donation/organtransplantation, as well as HLAs, Rh, and other blood groups, areassayed using protein, DNA, and/or RNA chip technology described hereinto detect antibodies, antigens, etc. in patient samples using thetechniques described herein.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

For example, kits for other specific symptom presentations include:Malaise/headache/myalgia/backache (Encephalitis) Kit, Conjunctivalhyperemia/lid edema/watery and/or mucopurulent discharge/preauricularlymphadenopathy (Conjuctivitis) Kit, Elevatedtemperature/tachycardia/increased respirations/leukocytosis/an impairedperipheral leukocyte response/oliguria (Septicemia) Kit, Septicemiaafter BMT/immunosupression Kit, Fever/chills/localized bone pain andtenderness/leukocytosis/bone deformation (Osteomyelitis) Kit, Tendernessof the urethra or the suprapubic area/temperature/shaking chills, nausea(Cystitis/pyelonephritis/urethritis) Kit, Tenderness of the pubic area,frequency, urgency, and dysuria in man (Prostatitis/epididimitis) Kit,Infertility, Ambigous Genitalia Kit, Hearing Loss (Hearing) Kit, Loss ofsight (Blindness) Kit, Mental Retardation Kit, Muscularweakness/pain/numbness (Neuromuscular) Kit, Muscularweakness/pain/numbness/mental retardation/tremor/(Neurological) Kit,Bone Deformation/pain (Bone) Kit, Cardiac dyspnea (Heart failure) Kit,Uremia (Kidney Failure) Kit, Malabsorption/weight loss (Gastro) Kit,Sinus pain/fever (Sinusitis) Kit, and Tropical Diseases Kit.

1. A method of determining a cause of one or more medical symptomsexhibited by a subject, the method comprising: (a) obtaining one or morebiological samples from the subject; (b) obtaining different probes ordifferent sets of probes, wherein each probe or set of probesselectively interacts with a target associated with a different knowncause of the one or more medical symptoms, and wherein the probesinclude at least (i) a first probe or set of first probes directed to afirst target, wherein the first target comprises a first marker for aninfectious agent known to cause the one or more medical symptoms; and(ii) a second probe or set of second probes directed to a second target,wherein the second target comprises a second marker for the infectiousagent; (c) applying the biological sample to the probes in the arrayunder conditions that enable all of the probes to selectively interactwith any targets in the biological sample; (d) detecting interactions;and (e) analyzing interactions to determine a cause of the one or moremedical symptoms. 2-34. (canceled)